Shupeng Yang

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.

In vitro and in vivo metabolite profiling of valnemulin using ultraperformance liquid chromatography-quadrupole/time-of-flight hybrid mass spectrometry.

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 vitro and in vivo metabolism of ochratoxin A: a comparative study using ultra-performance liquid chromatography-quadrupole/time-of-flight hybrid mass spectrometry

AbstractOchratoxin A (OTA) is a mycotoxin that frequently contaminates a wide variety of food and feedstuffs. The metabolism of OTA greatly affects fate and toxicity in humans and animals, because of its possible carcinogenic character (International Agency for Research on Cancer (IARC), group 2B). To completely characterize the metabolites of OTA, the metabolism of OTA in liver microsomes of rats, chickens, swine, goats, cows, and humans was investigated using ultra-performance liquid chromatography-quadrupole/time-of-flight hybrid mass spectrometry (UPLC-Q/TOF-MS). In addition, an in vivo comparative metabolism study of OTA was performed among rats and chickens after oral administration of OTA. As a result, a clear metabolic profile of OTA in different species was proposed, and a total of eight metabolites were identified, of which three hydroxylated metabolites at the phenylalanine moiety were discovered for the first time (preliminarily identified as 9′-OH-OTA, 7′-OH-OTA, and 5′-OH-OTA). Considerable amounts of 7′-OH-OTA were detected in different species’ liver microsomes, especially in chickens and humans. Moreover, the metabolism of OTA in chickens was elucidated for the first time in the present study. The 7′-OH-OTA proved to be the main metabolite in vitro and in vivo in chickens. Furthermore, the 4(S)-OH-OTA isomer was the major one, and 4(R)-OH-OTA the minor metabolite in chickens, which was different from others where 4R was the major. OTA undergoes metabolism via three different pathways, namely hydroxylation, dechlorination, and conjugation. The proposed metabolic pathways of OTA in various species provide the scientific community useful data for the toxicological safety evaluation of OTA among different species, and will further facilitate the food safety evaluation of OTA. Graphical abstractIn Vitro and in Vivo Metabolism of Ochratoxin A: A Comparative Study Using Ultra-Performance Liquid Chromatography-Quadrupole/Time-of-Flight Hybrid Mass Spectrometry

Unraveling the in vitro and in vivo metabolism of diacetoxyscirpenol in various animal species and human using ultrahigh-performance liquid chromatography-quadrupole/time-of-flight hybrid mass spectrometry.

AbstractDiacetoxyscirpenol (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 AbstractThe metabolism of diacetoxyscirpenol in farm animals and human

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.

Strategy for comparative untargeted metabolomics reveals honey markers of different floral and geographic origins using ultrahigh-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry

Honey discrimination based on floral and geographic origins is limited by the ability to determine reliable markers because developing hypothetical substances in advance considerably limits the throughput of metabolomics studies. Here, we present a novel approach to screen and elucidate honey markers based on comparative untargeted metabolomics using ultrahigh-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap). To reduce metabolite information losses during sample preparation, the honey samples were dissolved in water and centrifuged to remove insoluble particles prior to UHPLC-Q-Orbitrap analysis in positive and negative electrospray ionization modes. The data were pretreated using background subtraction, chromatographic peak extraction, normalization, transformation and scaling to remove interferences from unwanted biases and variance in the experimental data. The pretreated data were further processed using principal component analysis (PCA) and a three-stage approach (t-test, volcano plot and variable importance in projection (VIP) plot) to ensure marker authenticity. A correlation between the molecular and fragment ions with a mass accuracy of less than 1.0ppm was used to annotate and elucidate the marker structures, and the marker responses in real samples were used to confirm the effectiveness of the honey discrimination. Moreover, we evaluated the data quality using blank and quality control (QC) samples based on PCA clustering, retention times, normalized levels and peak areas. This strategy will help guide standardized, comparative untargeted metabolomics studies of honey and other agro-products from different floral and geographic origins.

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.

Optimization and application of parallel solid-phase extraction coupled with ultra-high performance liquid chromatography–tandem mass spectrometry for the determination of 11 aminoglycoside residues in honey and royal jelly

A robust and sensitive method of solid-phase extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established and performed for the simultaneous determination of eleven aminoglycosides (AGs) in royal jelly and honey. After sample extraction by a phosphate buffer containing trichloroacetic acid (TCA) and ethylenediaminetetracetic acid disodium salt (Na2EDTA), the extraction solution was subjected to a parallel solid-phase extraction for clean-up prior to the LC-MS/MS analysis. The same method was applied to analyze two completely different matrices, honey and royal jelly. Good sensitivity, repeatability, and recovery were obtained by using the mobile phase without an ion-pairing reagent such as heptafluorobutyric acid (HFBA) or sodium heptanesulfonate. The calibration curves of the honey and royal jelly samples exhibited a good linear response (R2 > 0.99) at six concentrations in the range of 10-1000 μg/mL. The limit of quantification (LOQ) of the AGs ranged from 10 to 25 μg/kg in the honey and from 12.5 to 25 μg/kg in the royal jelly. The recoveries of the AGs for the honey and royal jelly samples were in the range of 79.48% to 108.95% and 74.61% to 113.70% respectively and the relative standard deviations (RSDs) were between 1.23% and 9.59%, and between 1.51% and 9.98%, respectively. The proposed approach has been allowed in China as a reference method for the simultaneous determination of eleven AGs in honey and royal jelly.

Integrated immunoassay-based broad detection of multi-class mycotoxins

ABSTRACT 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.