Search

Fluorescence polarization immunoassay (FPIA) is a homogeneous and rapid analytical method that is suitable for high-throughput screening of large numbers of samples. However, FPIA typically suffers from lower sensitivity than the well-established enzyme-linked immunosorbent assay (ELISA), limiting its wide application as an analytical tool that can be run with trace levels of an analyte. Herein, a highly sensitive FPIA for detecting amantadine (AMD) in chicken is described. To achieve high sensitivity, nine chemical tracers of AMD that employ different fluoresceins, fluorescein derivatives, and haptens were synthesized and paired with four previously produced monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of FPIA was investigated and discussed. We found that the tracers with a linear and shorter bridge between adamantane and fluorescein generally provided higher sensitivity. After optimization, N'-(1-adamantyl) ethylenediamine (AEDA), an AMD structural analogue labeled with fluorescein isothiocyanate (FITC), achieved the lowest IC50 value (1.0 ng/ml) in the FPIA, which was comparable to that of the heterologous ELISA format that used the same mAb7G2. We also investigated the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. The developed FPIA was applied to chicken to detect AMD residue, demonstrating a limit of detection (LOD) of 0.9 µg/kg with recoveries of 76.5-89.3% and coefficients of variation (CVs) below 14.5%. These results show that the proposed FPIA is an efficient, accurate, and convenient method for the rapid screening of AMD residues in chicken. PRACTICAL APPLICATION: The fluorescence polarization immunoassay (FPIA) was developed to determine and quantify amantadine (AMD) in chicken samples with high sensitivity. This homogeneous method avoids coating and washing steps and may provide high-throughput AMD screening in chicken in 10 min with high accuracy and precision. FPIA can be used as a monitoring tool and contribute significantly to the rapid detection of AMD in chicken.

Phallotoxins, toxic cyclopeptides found in wild poisonous mushrooms, are predominant causes of fatal food poisoning. For the early and rapid diagnosis mushroom toxin poisoning, a highly sensitive and robust monoclonal antibody (mAb) against phallotoxins was produced for the first time. The half-maximum inhibition concentration (IC50) values of the mAb-based indirect competitive ELISAs for phallacidin (PCD) and phalloidin (PHD) detection were 0.31 ng mL−1 and 0.35 ng mL−1, respectively. In response to the demand for rapid screening of the type of poisoning and accurate determination of the severity of poisoning, colloidal gold nanoparticle (GNP) and time-resolved fluorescent nanosphere (TRFN) based lateral flow assays (LFA) were developed. The GNP-LFA has a visual cut-off value of 3.0 ng mL−1 for phallotoxins in human urine sample. The TRFN-LFA provides a quantitative readout signal with detection limit of 0.1 ng mL−1 in human urine sample. In this study, urine samples without pretreatment were used directly for the LFA strip tests, and both two LFAs were able to accomplish analysis within 10 min. The results demonstrated that LFAs based on the newly produced, highly sensitive, and robust mAb were able to be used for both rapid qualitative screening of the type of poisoning and accurate quantitative determination of the severity of poisoning after accidental ingestion by patients of toxic mushrooms.

In this study, a homologous multi-wavelength fluorescence polarization immunoassays (MWFPIA) for rapid determination of amantadine (AMD) and ribavirin (RVB) simultaneously was established. The MWFPIA could quantify AMD and RVB concentration simultaneously in 1 min. After optimisation, long-wavelength tracers AEDA-AF647 with monoclonal antibody (Mab) 1A12 and RVB-CP-EDF with polyclonal antibody (Pab) G412 were chosen for the development of MWFPIA. The limit of detections (LODs) of AMD and RVB were 1.7 and 1.0 μg/kg in chickens, and 17.6 and 10.4 μg/L in human serum, respectively. Recoveries of AMD and RVB in chickens were 61.2–94.9% and 87.3–90.2%, respectively with coefficient of variations (CVs) lower than 16.7%. For human serum, recoveries were 60.2–84.3% and 63.5–74.2% for AMD and RVB, respectively, and CVs was lower than 15.3%. The results indicated that the MWFPIA could be used for the monitoring of antiviral drugs with short analysis time, great accuracy and high throughput.

A multiplex immunochromatographic assay (ICA) based on dual-color fluorescent microspheres (FMs) as a sensitive label was developed for the first time. Two typical algae toxins, microcystin-LR (MC-LR) and okadaic acid (OA), were chosen as proof-of concept targets to evaluate the feasibility of this ICA format. Commercial red- and green-colored FMs were selected to couple with monoclonal antibodies as fluorescent probes. The use of dual-wavelength FMs as labels guaranteed a lower consumption of material strips, lower sample volume, and shorter reaction time without increasing the length of ICA strips. Under optimal conditions, the multiplex FM-ICA could be completed in 20 min and reached limits of detection for the simultaneous determination of MC-LR and OA in fish samples, which were 0.074 and 2.42 μg/kg, respectively. The developed technique was validated using artificially spiked and naturally contaminated fish samples. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used as confirmatory technique. In summary, this portable ICAs detection mode based on dual-wavelength FMs provided a reliable and sensitive on-site detection of multiple contaminants in food samples, which opens a new field for application of FMs in food safety.

Ribavirin (RBV) is an effective antiviral drug, whose use is prohibited in animal husbandry worldwide. In this work, a novel immunizing hapten of RBV, named Hapten 4, was designed by comparing the conformational and electronic properties of RBV and haptens based on computational chemistry. Hapten 4 was synthesized and conjugated with carrier proteins to produce monoclonal antibody (mAb). The obtained mAb 4C3 for RBV exhibited an IC50 value of 6.24 ng/ml in an indirect competitive enzyme-linked immunosorbent assay (icELISA) and displayed no cross-reaction with five other antiviral drugs, including amantadine. The applicability of the developed icELISA was verified in chicken, with a calculated limit of detection of 4.23 µg/kg. The recoveries in spiked chicken were 79.2%-107.3% with a coefficient of variation less than 15.9%. The results indicated that the produced antibody from the new hapten was reliable and would be useful for RBV screening in chicken. PRACTICAL APPLICATION: RBV is a broad-spectrum antiviral drug, which is commonly used illegally in poultry farms. A high-affinity mAb 4C3 against RBV was produced and used to develop icELISA with acceptable sensitivity and accuracy. The constructed icELISA has excellent performance for detecting RBV residues in chicken.

Lekethromycin, a new macrolide lactone, exhibits significant antibacterial activity. In this study, a reliable analytical ultrahigh-performance liquid chromatography electrospray ionization quadrupole Orbitrap high-resolution mass spectrometry (UPLC-ESI-Orbitrap-MS) method was established and validated for the detection of lekethromycin in rat plasma. After a simple acetonitrile (ACN)-mediated plasma protein precipitation, chromatographic separation was performed on a Phenomenex Luna Omega PS C18 column (30 &times, 2.1 mm i.d. particle size = 3 &mu, m) conducted in a gradient elution procedure using 0.5% formic acid (FA) in ACN and 0.5% FA in water as the mobile phase pumped at a flow rate of 0.3 mL/min. Detection was carried out under positive electrospray ionization (ESI+) conditions in parallel reaction monitoring (PRM) mode with observation of m/z 804.5580 &gt, 577.4056 for lekethromycin and 777.5471 &gt, 619.4522 for gamithromycin (internal standard, IS). The linear range was 5&ndash, 1000 ng/mL (r2 &gt, 0.99), and the lower limit of quantification (LLOQ) was 5 ng/mL. The intra- and inter-day precision (expressed as relative standard deviation, RSD) values were &le, 7.3% and &le, 6.3%, respectively, and the accuracy was &ge, 90% &plusmn, 5.3%. The mean extraction recovery RSD valWeue was &lt, 5.1%. Matrix effects and dilution integrity RSD values were &lt, 5.6% and &lt, 3.2%, respectively. Lekethromycin was deemed stable under certain storage conditions. This fully validated method was effectively applied to study the pharmacokinetics of lekethromycin after a single intravenous administration of 5 mg/kg in rats. The main pharmacokinetic parameters were T1/2&lambda, z, CL_obs and VZ_obs were 32.33 &plusmn, 14.63 h, 0.58 &plusmn, 0.17 L/h/kg and 25.56 &plusmn, 7.93 L/kg, respectively.

Antibody-based sensors are the most widely used for screening antibiotic residues in food samples. However, these sensors always suffer from cross-reacting antibiotics which are able to bind the employed antibodies in a manner similar to the target, thus resulting in inaccurate quantification. In this study, we developed a highly specific and sensitive chemiluminescence aptasensor for sulfamethazine (SMZ) by employing the supernormal selectivity of a new selected aptamer. Four SMZ-specific aptamers (Kd ranging from 79 to 274 nM) were selected in vitro by MB-SELEX with an intentional counter selection to avoid the recognition other sulfonamides, which is impossible to achieve in the corresponding procedure of antibody production. The binding mechanism of aptamers for SMZ was further analyzed and clarified with molecular simulation and docking. Under optimum conditions, the chemiluminescence aptasensor based on the highest affinity aptamer named SA07 provided a limit of detection of 0.92 ng/mL with negligible cross-reactivity with the 27 tested sulfonamides. The developed aptasensor was applied to milk samples, and achieved good accuracy and precision. These results demonstrated that the integration of an aptamer and chemiluminescence in a sensor platform is a promising method for the sensitive, specific, rapid and accurate detection of antibiotic residues of interest in food samples.

To increase information content per screen, a dual-wavelength, homologous and high-throughput fluorescence polarization immunoassay (DWFPIA) for simultaneous detection of total aflatoxins (AFs) and family zearalenones (ZENs) was developed. Aflatoxin B1 (AFB1) and zearalanone (ZAN) were labeled with different fluoresceins, and then combined with corresponding broad-specific antibodies to develop the DWFPIA. Under optimal conditions, the half maximal inhibitory concentrations of the FPIA in buffer were 2.68 μg/L and 4.08 μg/L for total four AFs (the weight ratio of AFB1: Aflatoxin B2: Aflatoxin G1: Aflatoxin G2 was 1: 1: 1: 1) and six ZEN analogs (the weight ratio of ZEN: ZAN: α-zearalenone: β-zearalenone: α-zearalanone: β-zearalanone was 1: 1: 1: 1: 1: 1) by mixing equal volumes (1: 1, v/v), respectively. The corresponding limit of detection values in maize flour samples were 4.98 μg/kg and 11.03 μg/kg, and the mean recoveries ranged from 78.6 to 103.6% with the coefficients of variation below 19.2%. The whole detection procedure, including sample preparation, took less than 30 min. Finally, the DWFPIA was applied to screen naturally contaminated maize flour samples, and the detection data were similar to those with HPLC-MS/MS.

Europium (Eu3+) and horse radish peroxidase (HRP) are highly promising biosensors due to its excellent physical and chemical properties. The two biosensors are widely applied in time-resolved fluoroimmunoassay (TRFIA) and chemiluminescence enzyme-linked immunosorbent assay, respectively. In this work, the two technologies were integrated with parallel input and parallel output (PI–PO) characters. Eu3+ was adopted as probe I in TRFIA for deoxynivalenol and de-epoxyldexoylnivalenol with a limit of detection (LOD) of 4.74 and 4.56 ng/mL, while HRP was performed as probe II in CL-ELISA for FB1 with an LOD of 0.088 ng/mL. Compared to the traditional individual assay, this method exhibited fast and broad detection characteristics by integrating two immunoassays into one.

Vitacoxib is an imidazole derivative and the novel COX-2 selective inhibitor to be marketed for veterinary use as nonsteroidal anti-inflammatory drugs. No analytical assay to quantify vitacoxib in equine plasma samples has been published to date. In the current study, we aim to develop and validate a brief, quick and sensitive UPLC–MS/MS method for quantification of vitacoxib in equine plasma samples. Plasma samples were precipitated with methyl tert-butyl ether. The Phenomenex column (Kinetex 50 × 2.1 mm i.d. particle size = 2.6 μm, C18, 100 A) at 25 °C was used in chromatographic separation with mobile phase consisting of acetonitrile and water (containing 0.1% formic acid) at flow rate of 0.4 mL/min. Vitacoxib and internal standard (IS, celecoxib) were detected under the multiple-reaction monitoring mode by mass spectrometer with ESI+ (m/z 347.9/269.03 for vitacoxib and m/z 382.0/362.0 for IS, respectively). The curve concentration range of was 0.5–500 ng/mL with a lower limit of quantification 0.5 ng/mL (r2 = 0.996309) in equine plasma samples. The selectivity, precision, recovery, accuracy, matrix effect and stability under various conditions were conformed to the acceptance requirements. Pharmacokinetic studies of vitacoxib in horses via oral administration (0.1 mg/kg) demonstrated that the procedure was fully validated and successfully. A meaningful basis for assessing the vitacoxib or clinical applications of vitacoxib to horse is provided in the present study.

After being incubated with animal and human liver microsomes, metabolites of phase I and II were investigated. A comparison was performed by ultrahigh performance liquid chromatography-quadrupole/time-of-flight coupled to mass spectrometry (UHPLC-Q/TOF). Consequently, a total of four phase I metabolites and three glucuronide binding metabolites of T-2 toxin were discovered. Although a significant metabolic difference was observed among six species, HT-2 toxin was the major product in all species. In addition, the in vivo metabolism of T-2 toxin after oral administration was also investigated in chickens, In total, 18 metabolites were detected, of which 13 were novel, to our knowledge, and reported for the first time. To elucidate the structures of these metabolites, besides accurate mass data from their MS and MS2 spectra, online hydrogen/deuterium (H/D) exchange technique was also carried out. These new metabolites were regarded as 3′-hydroxy-T-2 3-sulfate, 3′-hydroxy-HT-2 3-sulfate, 4′-hydroxy-HT-2, 3′,...

Multi-analyte immunoassays have attracted increasing attention due to their short assay times, low sample consumption, and reduced detection costs per assay. In this work, we describe a homologous and high-throughput multi-wavelength fluorescence polarization immunoassay (MWFPIA) for the multiplexed detection of mycotoxins. Three typical Fusarium mycotoxins, deoxynivalenol (DON), T-2 toxin and fumonisin B1 (FB1), were labeled with different dyes. Tracers and specific monoclonal antibodies (mAbs) were employed in the MWFPIA to simultaneously detect the three mycotoxins. Under optimal conditions, the limits of detection using MWFPIA were 242.0 μg kg(-1) for DON, 17.8 μg kg(-1) for T-2 toxin and 331.5 μg kg(-1) for FB1, providing sufficient sensitivity to meet the action levels of these three contaminants in maize as set by the European Union. The use of a methanol/water (2:3, v/v) mixture for sample pretreatment allowed recoveries ranging from 76.5-106.3%, with coefficients of variation less than 21.7%. The total time of analysis, including sample preparation, was less than 30 min. Twenty naturally contaminated maize samples were tested using MWFPIA and HPLC-MS/MS, with correlation coefficients (R(2)) of 0.97 for DON and 0.99 for FB1. By changing the targets of interest, homologous MWFPIA, a method with high sensitivity, a simple procedure and a short analysis time, can easily be extended to other chemical contaminants. Thus, MWFPIA represents a versatile strategy for food safety analysis.

Over the past few years, there has been a lack of progress in the quality of diethylstilbestrol (DES) antibodies used in immunoassay. In this study, a new immunizing hapten was designed for remarkably sensitive and specific antibody generation against diethylstilbestrol. By introducing a benzene ring instead of the traditional linear chain alkane as the hapten spacer, a more specific immune reaction was induced in the process of immunization. The developed polyclonal antibodies were characterized using the indirect competitive enzyme-linked immunosorbent assay (icELISA). Under optimized conditions, the half maximal inhibitory concentration (IC50) of the best polyclonal antibody was 0.14 ng/mL and it displayed low cross-reactions (CRs) with the structural analogs such as hexestrol (HEX) and dienestrol (DI). The molecular modeling and quantum chemical computation revealed that the lowest CR of the DES antibody to DI was mainly due to the huge three-dimensional conformational difference between DES and DI. Finally, a highly sensitive icELISA method based on the polyclonal antibody was developed for the determination of DES in shrimp tissue. The limit of detection (LOD) was as low as 0.2 μg/kg in shrimp and the recoveries in the spiked samples ranged from 83.4 to 90.8% with the coefficient of variation less than 13.8%. These results indicated that the use of an aromatic ring as the immunizing hapten spacer arm could be a potential strategy for the enhancement of anti-DES antibody sensitivity, and the established icELISA was applicable to the trace detection of DES in shrimp.

A biosensor that could simultaneously detect multi-analyte as many as in a single test is more favored when facing lots of samples for screening purpose. In such a biosensor, the recognition element with broad specificity and highly affinity is a key reagent for detection spectrum. Here we report a dihydropteroate synthase (DHPS) based biosensor for detecting 29 sulfonamides (SAs) in milk. The biosensor was established in a competitive format where HRP-labeled tracer and free SAs competitively binding to the limited amount of DHPS-DHPPP (6-hydroxymethyl-7,8-dihydropterin diphosphate) binary complex. Compared to other biosensors based on antibodies with the same objective, the current sensor employing DHPS provided not only highly sensitivity but also preponderant uniform affinities for all individual sulfonamide, which has never been achieved before in any immunosensor. However, the difference of recognition mechanism between DHPS and antibody remains unknown. To address this question, we deconstructed the variable region of two monoclonal antibodies produced by our group and then compared the intermolecular forces and key contacting amino acid residues of both DHPS and antibodies to several typical SAs with help of molecular modeling analysis. Results showed the orientation of the ligands in the binding site played a significant role during the recognition with proteins. The optimized assay provided a limit of detection (LOD) of 1.15–14.91 ng mL−1 and dynamic range was ranging from 1.54 to 126.85 ng mL−1 for 29 SAs. The developed biosensor finally was validated for five SAs in spiked milk with recovery values ranging from 72.7 to 129.3% and coefficients of variation less than 16.0%. The study showed that the biosensor based on DHPS make it a suitable screening method for the simultaneous determination of total SAs residues in food matrices.

Salbutamol (SAL) and ractopamine (RAC) have been illegally used to promote protein synthesis and to increase the feed conversion rate in livestock. However, the residues of SAL and RAC could cause potential hazards for human health. The Ministry of Agriculture of China banned the use of SAL and RAC as growth promoters. In this paper, we provide detailed information on developing a rapid and sensitive gel-based immunoassay for on-site screening of SAL and RAC residues in pork. The detection time was shortened to 20 min. The limits of detection were 0.5 μg/kg for both SAL and RAC by visual detection, whereas the quantitative gel-based immunoassay enabled the detection of SAL (0.051 μg/kg) and RAC (0.020 μg/kg) in spiked pork samples. The gel-based immunoassay showed promise as a multiplexed immunoassay for on-site surveilling of SAL and RAC residues in pork.

A rapid method for the liquid chromatography-tandem mass spectrometric determination of type A and B trichothecenes and their major metabolites in chicken meat, pork, chicken liver, and swine liver was developed. The analytes included T-2 toxin, HT-2 toxin, T-2 triol, neosolaniol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deepoxydeoxynivalenol, and nivalenol. The compounds were extracted from samples with acetonitrile/ethyl acetate (1:3, v/v) and then cleaned up using Oasis HLB cartridges. Analysis was carried out with ultraperformance liquid chromatography-tandem mass spectrometry. The mean recoveries of spiked samples ranged from 74.1% to 96.9% with intraday and interday relative standard deviations of less than 9.9% and 9.1%, respectively. The limit of detection and limit of quantitation ranged from 3.0 to 15.0 μg/kg and from 10.0 to 50.0 μg/kg, respectively. The proposed method has been successfully applied for analysis of real samples, with the primary results indicating that, compared to mycotoxins themselves, their metabolites are more likely to occur and be detectable in animal tissue foods.

Diacetoxyscirpenol (DAS), a Fusarium mycotoxin belonging to the trichothecene type A mycotoxins, is able to contaminate food and feed worldwide. Only limited information is available regarding the metabolism of DAS. The present study used ultrahigh-performance liquid chromatography-quadrupole/time-of-flight hybrid mass spectrometry (UHPLC-Q/TOF) to investigate the in vitro phase I and II metabolism of DAS by rat, chicken, swine, goat, cow, and human liver microsomes. An extensive metabolization profile of DAS has been observed. A total of seven phase I and three phase II metabolites of DAS were detected. Among the identified molecules, four phase I metabolites (8β-hydroxy-DAS, neosolaniol, 7-hydroxy-DAS, and its epimer) and two phase II metabolites (4-deacetyl-DAS-3-glucuronic acid and 4-deacetyl-DAS-4-glucuronic acid) were identified for the first time. These results indicate that the major metabolic pathways of DAS in vitro were hydrolyzation (M1-M3), hydroxylation (M4-M7), and conjugation (M8-M10). Qualitative differences in phase I and II metabolic profiles of DAS between the five animal species and human were observed. 4-Deacetyl-DAS was the primary metabolite from liver microsomes of all species, especially human. The in vivo metabolism of DAS in rats and chickens after oral administration of DAS was also investigated and compared. The major metabolites for rats and chickens were 4-deacetyl-DAS and 7-hydroxy-DAS. These results will help to gain a more detailed insight into the metabolism and toxicity of DAS among different animal species and human. Graphical Abstract The metabolism of diacetoxyscirpenol in farm animals and human.

In this work, three unreported immunizing haptens of ractopamine (RAC) were synthesized and used to produce highly sensitive and specific polyclonal antibody. The spacer arms of haptens for coupling to protein carrier were located on different position of RAC with different length. High affinity polyclonal antibodies were obtained and characterized in terms of titer and sensitivity by using enzyme-linked immunosorbent assay (ELISA). The best antibody employed in a heterologous competitive ELISA exhibited an IC50 value as low as 0.12ngmL(-1) and could not recognize other 10 β-agonists including clenbuterol and salbutamol. The heterologous competitive ELISA was preliminary applied to swine urine and the results showed the new antibody was sufficiently sensitive and specific, and potentially used for the detection of RAC at trace level in real samples.

Immunoglobulin Y (IgY) is derived from egg yolk and has been identified as a cheap and high-yield immunoreagent. The application of IgY in immunoassays for the detection of chemical contaminants in food samples has rarely been reported. In this work, we describe a rapid and sensitive fluorescence polarization immunoassay (FPIA) for valnemulin (VAL) using IgY which was produced using a previously prepared immunogen. Three fluorophore-labeled VAL tracers were synthesized and the sensitivity of the best tracer (VAL-DTAF) in the optimized FPIA with antibody IgY100 demonstrated an IC50 value of 12 ng mL(-1) in buffer. After evaluation of several extraction procedures, acidified acetonitrile was selected to extract VAL from swine tissue. The recoveries of VAL in spiked swine tissue at three levels (50, 100, and 200 μg kg(-1)) were higher than 79% with coefficients of variation (CVs) lower than 12%. The limit of detection (LOD) of the FPIA in swine tissue was 26 μg kg(-1) and was lower than the maximum residue limit (MRL) of VAL set by the European Union. The study showed that IgY could be a good substitute for IgG when developing a high-throughput assay for chemical residues.

A lateral flow immunoassay with silver staining for the simultaneous detection of fumonisin B1 (FB1) and deoxynivalenol (DON) in maize samples was reported. The assay was based on the competition between target mycotoxins and corresponding coating antigens immobilized on test lines for binding to limited gold nanoparticles (AuNPs)-labeled antibodies. The detection signal was further amplified by employment of silver staining on AuNPs. In the process, silver ions were catalyzed by AuNPs into metal silver that deposited on the surface of AuNPs, allowing not only the enlargement of particle dimensions of AuNPs but also a more distinguishable black coloration on the test zone. Under optimized conditions, the cut-off values of silver staining lateral flow immunoassay were 2.0 ng mL−1 for FB1 and 40 ng mL−1 for DON in buffer, which was improved at least 2 times in comparison to those of AuNPs-based method. The assay was further applied to detect FB1 and DON in naturally contaminated maize samples and a good agreement was found with the data obtained from HPLC-MS/MS.

The illegal use of β2-agonists in livestock production was previously detected by efficient methods based on mass spectrometry to control the residues of these drugs. Nevertheless, such methods still remain a challenging task for authorities who monitor these residues because the use of "cocktails" composed of mixtures of low amounts of several substances as well as the synthesis of new compounds of unknown structure prevent efficient prevention of illegal use of growth-promoting agents. Here, we outlined a metabolomics-based strategy for detecting the use of "cocktails" composed of mixtures of low amounts of three β2-agonists via urine profiling. Urine profiles of controls and swine treated with mixture of low amounts of three substances (clenbuterol, salbutamol, and ractopamine) were analyzed with ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The metabolic differences between controls and β2-agonists-treated groups were compared using multivariate data analysis. Fourteen metabolites were identified related with the β2-agonists treatment, while two co-biomarkers, 2-indolecarboxylic acid and fluorometholone acetate, either in single or "cocktails" of low-dose mixture of clenbuterol, salbutamol, and ractopamine, could be considered as diagnostic markers for the detection of illegal use of β2-agonists. The results of depletion study demonstrated that it is practical to use the markers for monitoring of β2-agonists.

Here we report a novel ultrasensitive surface-enhanced Raman scattering (SERS) immunosensor based on the flower-like gold nanoparticles (AuNFs) and magnetic bead separation for homogeneous detection of amantadine (AMD) in chicken just by one-step. The 5, 5′-dithiobis (2-nitrobenzoicacid) (DTNB) modified AuNFs and N-(1-adamantyl) ethylenediamine (AEDA) conjugated denatured BSA (AEDA-dBSA) was used as the SERS nanoprobe. And the capture probe was anti-AMD monoclonal antibody (mAbs)-functionalized magnetic beads (MNBs-mAbs). An immunoreaction occurred between free AMD and SERS nanoprobe for competing limited binding sites of MNBs-mAbs. This work combined inherent sensitive property of SERS with antibody-antigen highly specificity recognition for the AMD detection. The analytical results showed that the SERS-based immunosensor was sensitive, simple and reliable with a limit of detection (LOD) of 0.005 ng/mL for AMD, which were 2 orders of magnitude better than an enzyme-linked immunosorbent assay based on the same immunoreagents. Analysis of AMD-spiked chicken samples revealed that the developed immunosensor provided accepted recoveries ranging from 74.76%-89.34% with coefficient of variation less than 15.04%. This strategy represents a simple, reliable, and universal approach for detection of chemical contaminants in food samples.

A one-step generic chemiluminescence competitive direct enzyme immunoassay (CL-cdELISA) based on a single-chain variable fragment (scFv)–alkaline phosphatase (AP) fusion protein (CL-cdELISAscFv–AP) was developed. It was capable of detecting 20 targeted fluoroquinolones (FQs) in fish and shrimp matrices within 40 min below the maximum residue levels (MRLs). In the optimized generic assay, the scFv–AP fusion protein in combination with a norfloxacin–ovalbumin conjugate (NOR–OVA) showed 50% binding inhibition (IC50) at 0.15 ± 0.01 μg kg−1 for NOR in 0.01 M phosphate-buffered saline (PBS), indicating that it is seven times as sensitive as the corresponding competitive direct enzyme immunoassay (cdELISAscFv–AP), and the linear response range of the assay was extended from 0.04 to 1.08 μg kg−1. The limits of detection (LODs) of the assay for NOR were 0.017 μg kg−1 in shrimp and 0.018 μg kg−1 in fish, and the LODs inferred from the cross reactivity (CR) ranged from 0.013 μg kg−1 for ciprofloxacin (CIP) to 4.19 μg kg−1 for trovafloxacin (TRO); the recoveries of the three representative antibiotics norfloxacin, flumequine (FLU) and sarafloxacin (SAR) from spiked fish and shrimp samples varied from 72.50 to 118.50% and the mean coefficients of variation for the inter-assay and intra-assay were 6.4% and 9.2%, respectively. Further validation of CL-cdELISAscFv–AP with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that the assay was a reliable screening tool for the detection of FQs in fish and shrimp.

To explore differences of zearalenone (ZEN) metabolism between various species, phase I and II metabolism by liver microsomes of animals and human were investigated using ultra high-pressure liquid chromatography-quadrupole/time-of-flight mass spectrometry (UHPLC-Q/TOF MS). A total of 24 metabolites were identified, among which 12 were reported for the first time. Reduction, hydroxylation, and glucuronidation were the major metabolic pathways of ZEN, and significant differences in various species were also observed. Reduction was the main reaction in swine and human, whereas hydroxylation was predominant in rats, chickens, goats, and cows in in vitro systems. Furthemore, in vivo metabolism of ZEN in rats and chickens was investigated, and 23 and 6 metabolites were identified in each species, respectively. Reduction, hydroxylation, and glucuronidation were the major metabolic pathways in rats, while reduction and sulfation predominated in chickens. These results further enrich the biotransformation profile of ZEN, providing a helpful reference for assessing the risks to animals and humans.

In this study, 10 fluorescein-labeled ractopamine (RAC) derivatives (tracers) were synthesized and characterized to develop a rapid fluorescence polarization immunoassay (FPIA) for the detection of RAC in pork, using previously produced RAC polyclonal antibodies. The effect of the tracer structure on the sensitivity of the FPIA was investigated. The specificity of the FPIA was evaluated with 70 β-agonists and β-blockers. The FPIA showed a limit of detection of 0.56 μg kg−1 for RAC in pork, with recoveries ranging from 74.8% to 86.6% in spiked samples. The total analysis time, including sample pretreatment, was less than 1 h. The FPIA was used to screen 150 commercial pork samples for RAC residues and the results were consistent with those of an enzyme-linked immunosorbent assay (ELISA) and ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Our results demonstrate that the FPIA developed here is a rapid, accurate, and sensitive screening method for RAC residues in pork.

This manuscript describes the development of a sensitive, fast and easily-performed fluorescence polarization immunoassay (FPIA) for detection of microcystins (MCs) and nodularin-R (NOD) in water. MCs and NOD, the most widespread cyanobacterial toxin are hepatotoxins and tumor promoters, and their acute exposure may result in severe health problems in animals and humans. The fluorescein-based tracers were synthesized, and for the first time preparative high performance liquid chromatography (HPLC) was employed for their purification. Optimal tracers for the analysis were selected by evaluating the immunochemical activity. Under the optimal conditions, the achieved limits of detection (LODs) for MC-LR and NOD were 0.86 and 0.95 μg L−1, respectively, providing a sufficient sensitivity to meet the provisional guideline value recommended by the World Health Organization (WHO). An ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to confirm the accuracy and precision of the FPIA, and no obvious difference in recovery between these two methods was found. The correlation coefficients (R2) were higher than 0.968. The developed FPIA was easy-to-operate and could be completed within 10 min after simple filtration and adjustment of pH for water samples. The method can be easily extended for screening of other cyanotoxins, representing a versatile strategy for environmental sample analysis.

Microcystins (MCs) are cyclic heptapeptide toxins produced by various cyanobacterial genera that are toxic to both animals and humans. In this study, a novel strategy was proposed for the quantitation of nine MCs and Nodularin-R (NOD) in lake water using UHPLC-MS/MS under negative ionization mode, in which only centrifugation was employed during sample preparation. As a result, limits of quantification (LOQ) ranging from 0.05 to 0.1 μg/L for all studied compounds were obtained in water samples, which were lower than the results obtained using positive ionization mode. Additionally, validation was performed by spiking three different levels of MCs at 0.05 or 0.1, 0.5, 1.0 μg/L (n = 6). Recoveries ranged from 88.6% to 101.8%, and intraday and interday variability were lower than 12% and 14%, respectively, for all targeted compounds. Furthermore, the proposed method was applied to investigate microcystins contamination in fifty lake water samples collected in different regions in China. As a result, MC-LR, MC-RR, MC-YR, MC-WR, MC-LW, MC-LA, MC-LY, and MC-HilR were detected in lake water samples at trace level ranging from 0.06 to 0.37 μg/L. The obtained results indicated that it was necessary to monitor the presence of MCs in lake water, especially during regular cyanobacterial blooms during warmer months.

In animal husbandry, diazepam, chlorpromazine, and promethazine are illegally used to promote growth and increase weight, thus leading to food safety problems. Here, an easy and sensitive method was developed for simultaneous determination of these three drugs in lamb liver. Liver sample was extracted with 25 % ammonium hydroxide–ethyl acetate–chloroform (2:17:2, v/v/v) and cleaned up with an alumina N cartridge. The target compounds were quantified and identified by ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization tandem mass spectrometry (ESI/MS/MS) operated in multiple reaction monitoring mode. At the fortified levels of 0.5–5 μg kg−1, recoveries of all drugs in lamb liver were between 94.84 ± 9.78 % to 99.52 ± 11.25 % with 4.48–11.27 % of intra-day coefficient variations (CVs) and 5.72–12.00 % of inter-day CVs. Limit of detection and limit of quantification of the three drugs were 0.05–0.16 μg kg−1 and 0.15–0.50 μg · kg−1, respectively. The method was demonstrated to be reliable and sensitive.

The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA).

Valnemulin, a semisynthetic pleuromutilin derivative related to tiamulin, is broadly used to treat bacterial diseases of animals. Despite its widespread use, metabolism in animals has not yet been fully investigated. To better understand valnemulin biotransformation, in this study, metabolites of valnemulinin in in vitro and in vivo rats, chickens, swines, goats, and cows were identified and elucidated using ultraperformance liquid chromatography-quadrupole/time-of-flight hybrid mass spectrometry (UPLC-Q/TOF-MS). As a result, there were totally 7 metabolites of valnemulin identified in vitro and 75, 61, and 74 metabolites detected in in vivo rats, chickens, and swines, respectively, and the majority of metabolites were reported for the first time. The main metabolic pathways of valnemulin were found to be hydroxylation in the mutilin part (the ring system) and the side chain, oxidization on the sulfur of the side chain to form S-oxides, hydrolysis of the amido bond, and acetylization in the amido of the side chain. In addition, hydroxylation in the mutilin part was proposed to be the primary metabolic route. Furthermore, the results revealed that 2β-hydroxyvalnemulin (V1) and 8α-hydroxyvalnemulin (V2) were the major metabolites for rats and swines and S-oxides (V6) in chickens.

In this research, a reliable and rapid chemiluminescent ELISA (CL-ELISA) determination procedure was developed and validated for the detection of deoxynivalenol (DON) in rice. With the help of chemiluminescent substrate, this developed protocol exhibited a high-sensitive character with a limit of detection (LOD) value of 0.94 ng/mL while it was 6.12 ng/mL in our previous report with the conventional colorimetric ELISA procedure by using the same monoclonal antibody (mAb). The mAb used in this research was proved to tolerate no more than 20 % methanol. Therefore, after the extraction procedure with 20 % methanol, samples could be proceeded to take the detection steps directly without dilution by PBS. In spiked rice samples, mean recoveries ranged from 91.40 to 93.48 % with intra-day and inter-day relative standard deviation (RSD) less than 10.62 and 12.41 %, respectively. The results demonstrated that this developed high-sensitive CL-ELISA immunoassay was suitable for screening of DON in rice samples.

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.

The optimum conditions of an enzyme-linked immunosorbent assay (ELISA) in regard to different monoclonal antibodies (MAbs), assay format, immunoreagents, and several physicochemical factors (pH, salt, detergent, and solvent) were investigated to develop a broad-specific and sensitive immunoassay for detection of sulfonamides in milk samples. Two previously produced broad-specific MAbs, 4D11 and 4C7, and eight structurally different haptens conjugated with bovine serum albumin (BSA) were used as coating antigens in a competitive indirect ELISA (ciELISA). In addition, six hapten-HRP conjugates and the two MAbs were evaluated in a competitive direct ELISA. After optimization, a highly broad-specific and sensitive ciELISA for screening for sulfonamides was obtained based on MAb 4D11 and the BS-BSA heterologous-coating antigen, demonstrating a 50 % specific binding (IC50) for 22 sulfonamides at concentrations below 100 ng mL−1. This is the first report of an immunoassay that is capable of detecting more than 20 sulfonamides based on MAbs. The optimized ciELISA was used to quantify the five sulfonamides, SMZ, SDM, SQX, SMM, and SMX in spiked milk samples. Recoveries of 89–104.6 % and coefficients of variation of 11.9–19.1 % demonstrated the potential of the ciELISA to simultaneously monitor multiple sulfonamides in diluted milk samples without further purification steps.

This research produced a highly-specific and sensitive anti-T-2 toxin monoclonal antibody (mAb), and developed a rapid and sensitive competitive indirect enzyme-linked immunosorbent assay (ELISA) method for monitoring T-2 toxin in rice. The mAb showed a negligible cross-reactivity value (CR) to most of the mycotoxins, and it could specifically bind to T-2 toxin without other mycotoxins, including HT-2 toxin (CR value at 3.08%), which exhibited a similar structure to T-2 toxin. The limit of detection (LOD) value, measured by IC10, was 5.80 μg/kg. In spiked samples, mean recoveries ranged from 72.0% to 108.5% with intraday and interday variation less than 16.8 and 13.7%. This proposed protocol was significantly confirmed by a reliable ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method and significant correlation was obtained.

In this work, two novel haptens of tetracycline (TC) were synthesized and used to produce a polyclonal antibody for TC. A selective heterologous enzyme-linked immunosorbent assay (ELISA) using the novel antibody was optimized to exhibit an IC50 value as low as 0.7 ng mL−1 in buffer. The assay was sufficiently sensitive for the analysis of the maximum residue limit (MRL) of TC in milk (100 μg L−1). The analysis of TC in fortified milk samples indicated average recoveries of 84.9–111.8% with inter-assay CV values of 9.34–14.5% after 100-fold buffer dilution without any clean-up step or organic-solvent usage. The cross-reactivities of 17 other TC analogs were tested using the optimized ELISA, and a comparative molecular field analysis (CoMFA) was performed at the 3D level in a quantitative manner to explain the observed cross-reactivities. The results indicated that the CoMFA was a useful tool for understanding the recognition profiles of antibodies in competitive immunoassays for TCs.

A novel monoclonal antibody for orbifloxacin (ORB) was produced for the first time and used to develop a homogeneous fluorescence polarization immunoassay (FPIA) for ORB determination in milk. Three coating antigens and six fluorescent tracers were prepared, and evaluated by ELISA and FPIA methods, respectively. Heterogeneous tracer, LOM-BDF, was selected to develop the FPIA, providing high sensitivity and stability. Moreover, a new optimization strategy for tracer and Ab concentration was proposed by integrating Z′ factor into checkerboard titration, and Z′ factor >0.8 was considered as a prerequisite to ensure the robustness of quantitative FPIA. The optimized FPIA provided a detection limit of 3.9 ng mL−1 and IC50 of 24.5 ng mL−1 with an expected Z′ factor of 0.81. Saturated (NH4)2SO4 precipitation was applied for milk sample pre-treatment due to high tolerance to elevated-ionic strength in the FPIA. Mean recoveries of fortified milk ranged from 74.3 to 112% at the adding levels of 10, 20 and 40 ng mL−1. The results indicate that the developed FPIA is efficient and especially suitable for fast screening of ORB in milk.

To monitor spiramycin (SP) residue in milk, a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. This study described the preparation of three immunogens and the production of a high-affinity mAb. After optimization, the 50% inhibition concentration (IC50) for the developed icELISA was estimated as 0.97 ng/mL in the assay buffer, and the limit of detection and limit of quantitation were 2.51 and 4.40 μg/L in the milk matrix. The newly developed assay demonstrated negligible cross-reactivity with 15 other macrolide antibiotics, but not with kitasamycin (23.4%). The mean recoveries ranged from 81 to 103% for the spiked samples (5, 10, and 50 μg/L), and the coefficient of variation ranged from 5.4 to 9.6%. The icELISA was validated by LC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting SP residue in milk without requiring a cleanup process.

This paper describes a rapid one-step fluorescence polarization immunoassay (FPIA) for the simultaneous determination of multiple (fluoro)quinolone antibiotics (FQs) in food samples. Several fluorescent tracers were synthesized and evaluated in the FPIA method based on a broad-specificity of monoclonal antibodies toward FQs. The heterogeneous tracer, SAR-5-FAM, was considered as the optimal choice to prepare the immunocomplex single reagent, which allows a rapid and sensitive displacement reaction by addition of analytes. Optimized single-reagent FPIA exhibited broad cross-reactivities in the range of 7.8-172.2% with 16 FQs tested and was capable of determining most FQs at the level of maximum residue limits. Recoveries for spiked milk and chicken muscle samples were from 77.8 to 116%, with relative standard deviation lower than 17.4%. Therefore, this method could be applicable in routine screening analysis of multiple FQ residues in food samples.

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) method was established for the determination of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) in animal tissues and aquatic products. The analyte was extracted with 0.2 mol/L hydrochloric acid. The extract was cleaned up on a Bond Elut C18 cartridge. Then the eluate was collected and evaporated to dryness under nitrogen gas at 35 degrees C. The residue was redissolved in acetonitrile containing 0.1% (v/v) formic acid. The identification was performed by multiple reaction monitoring in positive electrospray ionization. The quantification was done by external standard method. The calibration curves showed good linearity within the range of 2-500 microg/L with the correlation coefficients (r2) greater than 0.990. The limits of detection (LODs) of MQCA in pork, swine liver, pig kidney, fish, prawn, and crab were 0.90, 1.51, 0.94, 1.04, 1.62 and 1.80 microg/kg, respectively; and the limits of quantification (LOQs) were 3.00, 5.02, 3.13, 3.46, 5.40 and 6.00 microg/kg, correspondingly. The recoveries of MQCA in animal tissues and aquatic products were 73.6%-89.0% at the spiked levels of 3-100 microg/kg. The intra-day relative standard deviations (RSDs, n = 5) were less than 15%, and inter-day RSDs (n = 3) were less than 20%. The results demonstrated that the sensitivity, accuracy, and precision were fit for the requirements of veterinary drug residue analysis.

This paper describes the preparation of reusable immunoaffinity columns and the development of an ultra-performance liquid chromatography tandem mass spectrometry method combined with immunoaffinity column clean-up (IAC-UPLC-MS/MS) for the determination of ochratoxin A (OTA) in cereals and feeds. The monoclonal antibody (mAb) was produced from a stable hybridoma cell line (4H10), which belongs to the immunoglobulin G1 (κ-light chain) isotype. A competitive indirect enzyme-linked immunosorbent assay was used to characterize the mAb. The concentrations causing 50 % inhibition of binding of mAb to OTA-ovalbumin by free OTA, ochratoxin B, and ochratoxin C were 1.29, 4.78, and 0.94 ng mL−1, respectively. The IAC-UPLC-MS/MS method offers a limit of quantification (LOQ, S/N >10) ranging from 0.5 to 1.0 μg kg−1 and a limit of detection (LOD, S/N >3) ranging from 0.2 to 0.3 μg kg−1 in cereal and feed samples. The IAC-UPLC-MS/MS method offers a good LOQ and LOD for OTA in cereal and feed samples. The accuracy and precision at this level fall within the EU regulatory limit. This methodology has been validated in four different matrices (millet, maize, soybean, and swine finisher diet) with highly satisfactory results and applied to the analysis of samples collected from the markets.

A gas chromatographic-tandem mass spectrometric method (GC-MS/MS) has been developed for sensitive and reliable detection of 8 organochlorine pesticide residues in wild L. chuanxiong and chestnut. The target compounds were extracted from both samples and simultaneously rudely cleaned up by water-acetone and dichloromethane. Then, the obtained dichloromethane phase was evaporated to dry, treated with H2SO4, centrifuged, evaporated and redissolved in 0.5 mL of benzene for GC-MS/MS detection. When spiked from 5 to 500 μg/kg, the average recoveries of organochlorine pesticides in two kinds of sample ranged from 74.2 to 110.9% with the intra-day RSD of 0.1 to 10.1% and the inter-day RSD of 0.7 to 10.5%. The limit of detection of eight compounds varied from 0.13 to 1.2 μg/kg in two kinds of samples.

Quinoxaline feed additives are antimicrobial growth promoters (AGPs); use three of them is permitted, and two of them are illegally used. It results in residue of quinoxaline AGPs and their metabolites in edible animal tissues, which are potentially harmful to human health. In order to effectively monitor the multiple residues of them in swine liver, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed based on polyclone antibody preparation. Protein conjugates were synthesized and immune to New Zealand white rabbit according to designed schemes. The effective antiserum with 50 % inhibiting concentration (IC50) value of 1.34 μg L−1 was obtained. A new synthesized quinoxaline with similar chemical structure to olaquindox but longer spacer arm was used as coating antigen. Cross-reactivity of other four quinoxaline AGPs and their eight metabolites was tested; seven of them have cross-reactivity over 10 %, with IC50 of 0.10–2.50 μg L−1. At the spike level of 1 to 100 μg kg−1 in swine liver, the recoveries of all compounds ranged from 80.14 % to 96.90 %, with the inter-day variation coefficient (CV) of 5.67–13.82 % and the intra-assay CV of 6.22–14.19 %. The limit of detection ranged from 0.03 ± 0.002 to 0.79 ± 0.05 μg L−1. Positive samples were determined by the ic-ELISA method and successfully confirmed by liquid chromatography-tandem mass spectrometry. The proposed ELISA is feasible for screening quinoxaline AGPs and their metabolites in swine liver.

Polyclonal and monoclonal antibodies against valnemulin, a new semi-synthetic antibiotic derivative of pleuromutilin administered to treat swine dysentery and pneumonia, were generated. To achieve high enzyme-linked immunosorbent assay sensitivity for valnemulin, several heterologous coating antigens were prepared and evaluated, differing in the length of the spacer arm and the conjugation site between valnemulin and carrier protein. After the optimisation of immuno-reagents dilution, the enzyme-linked immunosorbent assay, based on polyclonal antibody number 2 and one heterologous coating antigen, showed the highest sensitivity with an IC(50) value for valnemulin of 0.96 ng/mL in buffer. For spiked porcine liver, an extraction procedure with a mixture of acetonitrile and 0.01 M hydrochloric acid (40:60 v/v) was proposed and no further sample pre-treatment other than 10 times dilution of the extract was necessary prior to analysis, which gave recovery values ranging from 75.7% to 89.4%. The dynamic assay range and the limit of detection of the assay were 2.4-49.9 and 1.67 µg/kg for porcine liver, respectively. The assay was compared with a confirmation method based on LC-MS/MS by using valnemulin-treated samples, and a satisfactory correlation between both methods was observed.

Tiamulin is an antimicrobial widely used in veterinary practice to treat dysentery and pneumonia in pigs and poultry. However, knowledge about the metabolism of tiamulin is very limited in farm animals. To better understand the biotransformation of tiamulin, in the present study, in vitro and in vivo metabolites of tiamulin in rats, chickens, swine, goats, and cows were identified and elucidated using ultra-high performance liquid chromatography coupled to quadrupole/time-of-flight. As a result, a total of 26 metabolites of tiamulin, identified in vitro and in vivo, and majority of metabolites were revealed for the first time. In all farm animals, tiamulin undergoes phase I metabolic routes of hydroxylation in the mutilin part (the ring system), S-oxidation and N-deethylation on side chain, and no phase II metabolite was detected. Among these, 2β- and 8α-hydroxylation and N-deethylation were the main metabolic pathways of tiamulin in farm animals. In addition, we have put forward that 8a-hydroxy-tiamulin and 8a-hydroxy-N-deethyl-tiamulin could be hydroxylated into 8a-hydroxy-mutilin, the marker residue of tiamulin in swine. Furthermore, a significant interspecies difference was observed on the metabolism of tiamulin among various farm animals. The possible marker residues for tiamulin in swine were 8α-hydroxy-tiamulin, N-deethyl-tiamulin, and 8α-hydroxy-N-deethyl-tiamulin, which were consistent with the hypothesis proposed by the European Agency for the Evaluation of Medicinal Products. However, results in present study indicated that three metabolites (2β-hydroxy-tiamulin, N-deethyl-tiamulin, and 2β-hydroxy-N-deethyl-tiamulin) of tiamulin in chickens had larger yields, which implied that 2β-hydroxy-mutilin or N-deethyl-tiamulin was more likely to be regarded as the potential marker residue of tiamulin in chickens.

We describe a new strategy for using chemiluminescence resonance energy transfer (CRET) by employing hapten-functionalized quantum dots (QDs) in a competitive immunoassay for detection of sulfamethazine (SMZ). Core/multishell QDs were synthesized and modified with phospholipid-PEG. The modified QDs were functionalized with the hapten 4-(4-aminophenyl-sulfonamido)butanoic acid. The CRET-based immunoassay exhibited a limit of detection for SMZ of 9 pg mL–1, which is >4 orders of magnitude better than a homogeneous fluorescence polarization immunoassay and is 2 orders of magnitude better than a heterogeneous enzyme-linked immunosorbent assay. This strategy represents a simple, reliable, and universal approach for detection of chemical contaminants.

Fumonisin B₁ (FB₁) is a mycotoxin, mainly produced by Fusarium fungi and present in food and feed. It causes harmful effects on human and animal health. Therefore, it is necessary to develop sensitive and reliable screening methods. In this study, a highly sensitive monoclonal antibody (MAb) against FB₁, clone 2D7, was produced, and the 50% inhibition concentration (IC₅₀) of the MAb was 2.2 ng/mL in buffer. The MAb showed high cross-reactivity with fumonisin B₂ (FB₂), and negligible cross-reactivity with other mycotoxins. A sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this MAb was developed for the determination of FB₁ in maize. In spiked samples (100, 200 and 500 μg/kg), the average recoveries ranged from 78 ± 11 to 107 ± 4%, and the coefficient of variation ranged from 3 to 15%. The limit of detection of the icELISA was 5.4 μg/kg. This method was compared to liquid chromatography tandem mass spectrometry (LC-MS/MS) using naturally contaminated samples, and the correlation coefficient was above 0.82. These results show the reliability of the icELISA method for the determination of FB₁ in maize.

This research paper presents a comparative study of three formats of immunoassays, including conventional enzyme-linked immunosorbent assay (ELISA), one-step ELISA, and reverse enzyme-linked immunosorbent assay (reELISA), for the determination of T-2 toxin (T-2) and HT-2 toxin (HT-2). This comparative study was performed with regard to specificity, sensitivity, matrix effect, accuracy, and precision. Among the three procedures, reELISA exhibited the best sensitivity with limits of detection (LOD) at 3.04 and 2.79 ng mL−1 for T-2 and HT-2. For the other parameters, there were no apparent differences. Good recoveries in negative spiked samples at concentrations of 50, 200, and 500 ng mL−1 were obtained, and the values were 67.9–112.9% for conventional ELISA, 60.8–108.8% for one-step ELISA, and 62.7–88.1% for reELISA. The relative standard deviation (RSD) for each procedure was less than 10.3%, 14.0%, and 11.5%, respectively. Among all three methods, the one-step protocol was the most time efficient, and the...

A rapid and sensitive method based on competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed and validated for the detection of DON (deoxynivalenol) in cereals with the confirmation of the reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Within this method, the IC50 (half maximal inhibitory concentration of a substance) of the DON-specific monoclonal antibody (MAb) we produced was 61.10 ng/mL. The limit of detection (LOD) value, measured by IC10, was 6.12 ng/mL, which is below the maximum residue levels (MRLs) established for DON in various cereals. In spiked samples, the recoveries ranged from 79.3% to 115.2% for maize and from 74.3% to 118.3% for wheat. This method was compared with the UPLC-MS/MS method by testing four concentrations. The correlation coefficient for the two methods was 0.9884 in a linear-regression analysis. The results illustrated the reliability of the ciELISA method for the determination of cereal samples.

A rapid and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of cyadox (CYX) and its three major metabolites, quinoxaline-2-carboxylic acid (QCA), 1-desoxycyadox (1-DCYX) and 1,4-bisdesoxycyadox (BDCYX) in fish. Samples were extracted with acetonitrile–0.5 M hydrochloric acid (9 + 1) and cleaned up using Oasis MAX cartridges. The total time taken for separation by UPLC was less than 5 min. The four target compounds were then determined by ESI-MS/MS. At the fortified levels of 2–50 μg kg−1 in fish, recoveries of all the compounds ranged from 80.2% to 88.2%, with a relative standard deviation of 6.8 to 14.6%. Limits of detection for CYX, QCA, 1–DCYX and BDCYX were 0.52, 1.07, 0.69 and 0.38 μg kg−1, respectively.

A sensitive and reliable method was developed and validated for the analysis of ractopamine in swine tissues. The sample preparation procedure was based on hydrolysis overnight, extraction with ethyl acetate, and solid-phase extraction cleanup. The target compound was determined by ultra-performance liquid chromatography coupled with tandem mass spectrometry. The average recoveries ranged between 93.3% and 106.5%, with relative standard deviations of 7.2–12.8%. The detection and quantification limits were 0.2 and 0.5 μg/kg, respectively. The depletion profile of ractopamine was studied in healthy pigs after oral administration of feed containing 20 mg/kg ractopamine for 30 consecutive days. Ractopamine residues were still detected 11 days post-medication in all tissues examined with the exception of muscle. The highest ractopamine level was found in lung tissue. The developed method has been successfully applied to the depletion study of ractopamine in swine tissues.