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.Keywords: comparative metabolism; farm animals; in vivo; liver microsomes; tiamulin; UHPLC−Q/TOF;

•This is the first study on quantifying vitacoxib in equine plasma.•This UPLC–MS/MS method with simple, rapid and sensitive was established.•The method was first successfully applied to a pharmacokinetic study after oral administrations of vitacoxib in horses.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 Å) 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.

•We have reported three new immunizing hapten of ractopamine.•Several highly sensitive and specific polyclonal antibodies were produced.•All antibodies were more sensitive in heterologous coating antigen ELISA.•One ELISA method was applied to swine urine with acceptable recovery and precision.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 0.12 ng mL−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.

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.

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.

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.

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.

In the present study, metabolites of T-2 toxin in in vivo and in vitro systems of Wistar rats were identified and elucidated by ultraperformance liquid chromatography–quadrupole/time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS). Expected and unexpected metabolites were detected by MetabolynxXS software, which could automatically compare MSE data from the sample and control. A total of 19 metabolites of T-2 toxin were identified in this research, 9 of them being novel, which were 15-deacetyl-T-2, 3′-OH-15-deacetyl-T-2, 3′,7-dihydroxy-T-2, isomer of 3′,7-dihydroxy-T-2, 7-OH-HT-2, isomer of 7-OH-HT-2, de-epoxy-3′,7-dihydroxy-HT-2, 9-OH-T-2, and 3′,9-dihydroxy-T-2. The results showed that the main metabolic pathways of T-2 toxin were hydrolysis, hydroxylation, and de-epoxidation. In addition, the results also revealed one novel metabolic pathway of T-2 toxin, hydroxylation at C-9 position, which was demonstrated by the metabolites 9-OH-T-2 and 3′,9-dihydroxy-T-2. In addition, hydroxylation at C-9 of T-2 toxin was also generated in in vitro of liver systems. Interestingly, several metabolites of hydroxylation at C-7 of T-2 toxin were also detected in in vivo male Wistar rats, but they were not found in in vivo female rats and in in vitro systems of Wistar rats.

The gene encoding the dihydropteroate synthase (DHPS) of Streptococcus pneumonia has been cloned, sequenced, and expressed in Escherichia coli. The protein has been purified and used to develop a novel microplate assay for the detection of sulfonamides. The assay was based on the competition between sulfonamides and horseradish peroxidase (HRP)-labeled sulfonamide derivative, 4-(4-aminobenzenesulfonylamino) benzoic acid (CS) for the immobilized protein. Under optimized conditions, nine sulfonamides and p-aminobenzoic acid (PABA) could be detected below 100 ng/ml and 28 sulfonamides used in the study could be detected with IC50 values ranging from 426 to 50,000 ng/ml. It is concluded that this method offers a robust and rapid alternative to other methods for the screening of sulfonamides.

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.

In this work, metabolites of cyadox (CYX) produced by liver microsomes, primary hepatocytes and intestinal microflora systems of rat, chicken and swine were identified and elucidated using ultra-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS). Expected and unexpected metabolites were found out using MetabolynxXS software by automatically comparing MSE data from the sample and control. The metabolites were reliably characterized by accurate MS/MS spectra and their different fragmentation pathways. In total twenty-four metabolites were identified in the three in vitro systems, fifteen of which were novel. The results revealed the main metabolic pathways of CYX were N→O group reduction, hydroxylation and hydrolysis reactions. Besides, methylation and acetylation reactions which represented phase II drug biotransformations were recognized in the in vitro systems of chicken firstly. It also showed that the metabolic capacity and rapidity of chicken and swine was greater than rat, with some differences in metabolic fate and metabolite varieties. This work contributes to the comprehensive understanding of in vitro metabolism of CYX, and will provide an important basis for further in vivo metabolism study and toxicological safety evaluation.

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 new ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of mequindox (MEQ) and its two metabolites (1-desoxymequindox and 1, 4-bisdesoxymequindox, 1-DMEQ and BDMEQ) in swine liver. This method was validated on the basis of Commission Decision 2002/657/EC. Target analytes were extracted from swine liver with acid acetonitrile, purified by an Oasis MAX cartridge, and separated by UPLC. The chromatography total time was less than 8 min with gradient elution of 0.1% formic acid in methanol. Data acquisition was carried out by electrospray ionization tandem mass spectrometry (ESI/MS/MS) operated in a multiple reaction monitoring mode. At fortified levels from 2 to 100 μg kg−1 in swine liver, recoveries of target analytes ranged from 80% to 85% with the intra-day coefficient of variation (CV) being ≤ 14.48% and inter-day CV ≤ 14.53%, respectively. The limit of detection (LOD) ranged from 0.58 to 1.02 μg kg−1 and the limit of quantification (LOQ) range was 1.93 to 3.40 μg kg−1 for each analyte. The result indicated that this method was specific, sensitive, and suitable for the quantification and conformation of MEQ and its two metabolites in swine liver.

A rapid and sensitive method based on chemiluminescent competitive indirect ELISA (CL-ciELISA) was developed for the screening of gentamicin (GEN) in milk. The cross-reactivity (CR) of gentamicin, sisomicin (SIS), neomycin (NEO), kanamycin (KAN), neamine (NEA), streptomycin (STR), apramycin (APR) and spectomycin (SPT) with this GEN-specific monoclonal antibody (MAb) produced in this experiment were 100, 3.2, <0.1, <0.1, <0.1, <0.1, <0.1, and <0.1%, respectively. In this method, IC50 (half maximal inhibitory concentration of a substance) of the MAb was 0.69 ng mL−1 and the limit of detection (LOD), measured by IC10, was 0.06 ng mL−1. This CL-ciELISA method with luminol solution as a substrate was 10 times more sensitive compared to the colorimetric-ciELISA. In spiked milk samples, the mean recoveries ranged from 85.6–97.9% with intra-day and inter-day variation less than 7.9 and 7.98%, respectively. The results illustrated that this procedure is applicable for screening gentamicin in milk.

A reliable and sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of 5 trichothecenes (deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 toxin, HT-2 toxin) in rice was developed. These target analytes were extracted by acetonitrile/water (84:16, v/v), and further purified with solid phase extraction Oasis HLB cartridge. In spiked samples, the mean recoveries ranged from 71.2–102.5% with intra-day and inter-day coefficients of variation less than 11.7 and 12.9%, respectively. Limits of detection (LOD) and quantification (LOQ) were in the range of 0.2–3 μg kg−1 and 0.5–10 μg kg−1, respectively. The results illustrate that this sensitive procedure is applicable for the determination of these trichothecenes in rice.

This review focuses on the toxicity and metabolism of T-2 toxin and analytical methods used for the determination of T-2 toxin. Among the naturally occurring trichothecenes in food and feed, T-2 toxin is a cytotoxic fungal secondary metabolite produced by various species of Fusarium. Following ingestion, T-2 toxin causes acute and chronic toxicity and induces apoptosis in the immune system and fetal tissues. T-2 toxin is usually metabolized and eliminated after ingestion, yielding more than 20 metabolites. Consequently, there is a possibility of human consumption of animal products contaminated with T-2 toxin and its metabolites. Several methods for the determination of T-2 toxin based on traditional chromatographic, immunoassay, or mass spectroscopy techniques are described. This review will contribute to a better understanding of T-2 toxin exposure in animals and humans and T-2 toxin metabolism, toxicity, and analytical methods, which may be useful in risk assessment and control of T-2 toxin exposure.

A high-performance liquid chromatography–diode array detector–tandem mass spectrometry (HPLC–DAD–MS/MS) method was developed for simultaneous determination of melittin and apamin in crude bee venom lyophilized powder (CBVLP) as the traditional Chinese medicine possessing specific biological activity. Melittin and apamin were extracted with pure water from CBVLP samples followed by HPLC–DAD–MS/MS analysis. The method was validated to demonstrate its selectivity, linearity, limit of quantification (LOQ), intraday precision, interday precision, accuracy, recovery, matrix effect, and stability. The assay was linear over the concentration ranges of 1–100 and 0.2–25 μg/ml with limit of quantifications (LOQs) of 1.0 and 0.3 μg/ml for melittin and apamin, respectively. The precision results were expressed as coefficients of variation (CVs), ranging from 2.2% to 11.4% for intraday repeatability and from 3.2% to 13.1% for interday intermediary precision. The concentrations of endogenous melittin and apamin in CBVLP samples ranged from 46% to 53% and from 2.2% to 3.7% of dry weight, respectively. This rapid, simple, precise, and sensitive method allowed the simultaneous determination of melittin and apamin to evaluate authenticity and quality of CBVLP samples.

A time-resolved fluoroimmunoassay (TR-FIA) for determination of chloramphenicol (CAP) in shrimp and chicken muscle was developed. The method was based on a direct competitive immunoassay using europium-labeled anti-CAP monoclonal antibody (MAb) and CAP-ovalbumin as coated antigen. The limit of detection was 0.05 ng g−1 and limit of quantification was 0.1 ng g−1. Recoveries ranged from 101.2 to 112.5% for shrimp and 104.9 to 115.3% for chicken muscle at spiked levels of 0.1–5 ng g−1, with intra-assay and inter-assay variations 8.7–14.6 and 9.6–17.8%, respectively. The results obtained by the TR-FIA and ELISA correlated well. The established TR-FIA was validated for the determination of incurred shrimp samples and confirmed by gas chromatography with microcell electron capture detector (GC-μECD).

A new ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of mequindox (MEQ) and its two metabolites (1-desoxymequindox and 1, 4-bisdesoxymequindox, 1-DMEQ and BDMEQ) in swine liver. This method was validated on the basis of Commission Decision 2002/657/EC. Target analytes were extracted from swine liver with acid acetonitrile, purified by an Oasis MAX cartridge, and separated by UPLC. The chromatography total time was less than 8 min with gradient elution of 0.1% formic acid in methanol. Data acquisition was carried out by electrospray ionization tandem mass spectrometry (ESI/MS/MS) operated in a multiple reaction monitoring mode. At fortified levels from 2 to 100 μg kg−1 in swine liver, recoveries of target analytes ranged from 80% to 85% with the intra-day coefficient of variation (CV) being ≤ 14.48% and inter-day CV ≤ 14.53%, respectively. The limit of detection (LOD) ranged from 0.58 to 1.02 μg kg−1 and the limit of quantification (LOQ) range was 1.93 to 3.40 μg kg−1 for each analyte. The result indicated that this method was specific, sensitive, and suitable for the quantification and conformation of MEQ and its two metabolites in swine liver.

A reliable and sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of 5 trichothecenes (deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 toxin, HT-2 toxin) in rice was developed. These target analytes were extracted by acetonitrile/water (84:16, v/v), and further purified with solid phase extraction Oasis HLB cartridge. In spiked samples, the mean recoveries ranged from 71.2–102.5% with intra-day and inter-day coefficients of variation less than 11.7 and 12.9%, respectively. Limits of detection (LOD) and quantification (LOQ) were in the range of 0.2–3 μg kg−1 and 0.5–10 μg kg−1, respectively. The results illustrate that this sensitive procedure is applicable for the determination of these trichothecenes in rice.

A rapid and sensitive method based on chemiluminescent competitive indirect ELISA (CL-ciELISA) was developed for the screening of gentamicin (GEN) in milk. The cross-reactivity (CR) of gentamicin, sisomicin (SIS), neomycin (NEO), kanamycin (KAN), neamine (NEA), streptomycin (STR), apramycin (APR) and spectomycin (SPT) with this GEN-specific monoclonal antibody (MAb) produced in this experiment were 100, 3.2, <0.1, <0.1, <0.1, <0.1, <0.1, and <0.1%, respectively. In this method, IC50 (half maximal inhibitory concentration of a substance) of the MAb was 0.69 ng mL−1 and the limit of detection (LOD), measured by IC10, was 0.06 ng mL−1. This CL-ciELISA method with luminol solution as a substrate was 10 times more sensitive compared to the colorimetric-ciELISA. In spiked milk samples, the mean recoveries ranged from 85.6–97.9% with intra-day and inter-day variation less than 7.9 and 7.98%, respectively. The results illustrated that this procedure is applicable for screening gentamicin in milk.

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.