Yanshen Li

T-2 Toxin, a Trichothecene Mycotoxin: Review of Toxicity, Metabolism, and Analytical Methods

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.

Identification of the major metabolites of quinocetone in swine urine using ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Quinocetone (QCT), 3-methyl-2-cinnamoylquinoxaline-1,4-dioxide, is a quinoxaline-N,N-dioxide used in veterinary medicine as a feed additive. QCT is broadly used in China to promote animal growth, but few studies have been performed to reveal the metabolism of QCT in animals until now. In the present study, the metabolites of QCT in swine urine were investigated using ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS). Multiple scans of metabolites in MS and MS/MS modes and accurate mass measurements were performed simultaneously through data-dependent acquisition. Most measured mass errors were less than +/-5 mDa for both protonated molecules and product ions using external mass calibration. The structures of metabolites and their product ions were easily and reliably characterized based on the accurate MS(2) spectra and known structure of QCT. As expected, extensive metabolism was observed in swine urine. Thirty-one metabolites were identified in swine urine, most of which were reported for the first time. The results reveal that the N-O group reduction at position 1 and the hydroxylation reaction occurring at the methyl group, the side chain or on the benzene ring are the main metabolic pathways of quinocetone in swine urine. There was abundant production of 1-desoxyquinocetone and hydroxylation metabolites of 1-desoxyquinocetone. The proposed metabolic pathway of quinocetone in vivo can be expected to play a key role in food safety evaluations.

High Specific Monoclonal Antibody Production and Development of an ELISA Method for Monitoring T-2 Toxin in Rice

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.

Development of a rapid competitive indirect ELISA procedure for the determination of deoxynivalenol in cereals

Abstract 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.

Determination of deoxynivalenol in cereals by immunoaffinity clean-up and ultra-high performance liquid chromatography tandem mass spectrometry.

An immunoaffinity column (IAC) was prepared with a new deoxynivalenol (DON) monoclonal antibody and used as a clean-up tool before ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis of DON in cereals. The developed IAC clean-up method showed high recoveries for DON. They ranged from 61% to 103% in wheat, rice, and millet with intra-day and inter-day variations below 19% and 17%, respectively. The column capacity was 2.86μg DON per mL of gel, and it maintained above 0.68μg/mL of gel after 10 cycles of usage at 2 days intervals. The limit of detection (LOD) and limit of quantification (LOQ) were 0.3 and 0.8μg/kg, respectively. Twenty-one out of 40 analyzed commercial cereal samples were positive at DON concentrations from 7 to 534μg/kg.

Development of a chemiluminescent competitive indirect ELISA method procedure for the determination of gentamicin in milk

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.

Comprehensive Analysis of Tiamulin Metabolites in Various Species of Farm Animals Using Ultra-High-Performance Liquid Chromatography Coupled to Quadrupole/Time-of-Flight

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.

Determination of T-2 Toxin and HT-2 Toxin in Milk: A Comparison of Three Formats of Immunoassays

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 reELISA procedure exhibited the best sensitivity.

Metabolic Profile of Zearalenone in Liver Microsomes from Different Species and Its in Vivo Metabolism in Rats and Chickens Using Ultra High-Pressure Liquid Chromatography-Quadrupole/Time-of-Flight Mass Spectrometry

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.

Simultaneous determination of type-A and type-B trichothecenes in rice by UPLC-MS/MS

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.