Zheng Han

A liquid chromatography tandem mass spectrometry method for simultaneous determination of acid/alkaline phytohormones in grapes

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of five acid/alkaline phytohormones, i.e., indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthylacetic acid (NAA), gibberellic acid (GA(3)) and isopentenyladenine (2IP), in grapes was developed. After optimization, the samples were extracted with methanol containing 1% formic acid and purified by Oasis HLB SPE cartridges. The analytes were separated on a Thermo Hypersil Gold column (100 mm×2.1 mm, 3.0 μm) with water and acetonitrile, then determined with Thermo tandem quadrupole mass spectrometer operating in negative electro-spray ionization using selected reaction monitoring (SRM) mode. The established method was further validated by determining the linearity (R² ≥ 0.9990), average recovery (82.5-105.4%), sensitivity (0.05-1.00 ng mL⁻¹), precision (RSD ≤1 3.0%) and stability (RSD ≥ 82.0%). Finally, the application of the approach proposed to thirty grape samples convinced its desirable performance for rapid analysis of multiclass phytohormones, supporting its sufficient capability for multiresidue analyses or other analytical system targeting phytohormones in agriculture field.

A reliable liquid chromatography–tandem mass spectrometry method for simultaneous determination of multiple mycotoxins in fresh fish and dried seafoods

A reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of nine mycotoxins, i.e., aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), ochratoxin A (OTA), zearalenone (ZEN), T-2 toxin, HT2 toxin and deoxynivalenol (DON), in fresh fish (muscle and entrails) as well as dried seafoods. Special focus was given to sample pretreatment which is crucial for an accurate and reliable analytical method. With regards to the high complexity of the matrices, extraction solvent, time, and temperature as well as clean-up cartridges were optimized to improve extraction efficiency and reduce matrix effects. The optimum procedure included ultrasound-assisted extraction with acetonitrile/water/acetic acid (79/20/1, v/v/v) at 40 °C for 30 min, defatting with n-hexane and purification by Oasis HLB cartridges. The method was further validated by determining the linearity (R(2) ≥ 0.9989), sensitivity (limit of detection ≤ 2 μg/kg, limit of quantitation ≤ 3 μg/kg), recovery (72.2-119.9%) and precision (≤ 18.3%) in muscle and entrails of fresh crucian carp (Carassius carassius) as well as dried fish products. The method was proven to be suitable for its intended purposes. Mycotoxins of OTA, ZEN and AFB2 have been found in fresh fish and dried seafoods for the first time.

Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster

ABSTRACT The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.

An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS-NRPS is necessary for synthesis of the 2-pyridones, leporins.

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.

Simultaneous determination of major type B trichothecenes and deoxynivalenol-3-glucoside in animal feed and raw materials using improved DSPE combined with LC-MS/MS.

A simple and reliable method for simultaneous determination of deoxynivalenol-3-glucoside and major type B trichothecenes (deoxynivalenol, nivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol and deepoxy-deoxynivalenol) in animal feed and raw materials has been developed and validated in this study. The method was based on an improved dispersive solid-phase extraction (DSPE) followed by analysis using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Also, matrix-matched calibration curve (R(2)>0.99) was employed to minimize matrix effects and ensure accurate quantification. The recoveries during sample preparation process (including extraction and clean-up) ranged from 79.03% to 118.39%, with intra-day and inter-day relative standard deviation lower than 20% for all the analytes. The limit of quantification ranged from 5.0 μg/kg for deoxynivalenol to 13.6 μg/kg for fusarenon X. The validated method was successfully applied to the analysis of animal feed and corn. The pilot study showed that 37 out of 41 samples were contaminated with deoxynivalenol-3-glucoside at the levels of 6.0-121.0 μg/kg. Most of the type B trichothecenes were also found with the exception of fusarenon X, at the contaminated levels of 10.0-1,382 μg/kg. To the best of our knowledge, this was the first scientific report on the co-occurrence of masked deoxynivalenol and type B trichothecenes in animal feed and raw materials.

A quick, easy, cheap, effective, rugged, and safe sample pretreatment and liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of 33 mycotoxins in Lentinula edodes.

Lentinula edodes, one of the most cultivated edible fungi in the world, are usually neglected for mycotoxins contamination due to the initial thinking of its resistance to mycotoxingenic molds. In the present study, a sensitive and reliable liquid chromatography with tandem mass spectrometry method was developed for the simultaneous quantification of 33 mycotoxins in L. edodes. Targeted mycotoxins were extracted using a quick, easy, cheap, effective, rugged, and safe procedure without any further clean-up step, and analyzed by liquid chromatography with tandem mass spectrometry on an Agilent Poroshell 120 EC-C18 column (100 × 3 mm, 2.7 μm) with a linear gradient elution program using water containing 5 mM ammonium acetate and methanol as the mobile phase. After validation by determining linearity (R(2) > 0.99), sensitivity (LOQ ≤ 20 ng/kg), recovery (73.6-117.9%), and precision (0.8-19.5%), the established method has been successfully applied to reveal the contamination states of various mycotoxins in L. edodes. Among the 30 tested samples, 22 were contaminated by various mycotoxins with the concentration levels ranging from 3.3-28,850.7 μg/kg, predicting that the edible fungus could be infected by the mycotoxins-producing fungi. To the best of our knowledge, this is the first report about real mycotoxins contamination in L. edodes.

Combinatorial approach of LC–MS/MS and LC–TOF-MS for uncovering in vivo kinetics and biotransformation of ochratoxin A in rat

A combinatorial platform of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography coupled with time of flight mass spectrometry (LC-TOF-MS) has been developed to investigate the in vivo kinetics and biotransformation of ochratoxin A (OTA) in rats. The stable isotope dilution LC-MS/MS method was first validated by determining the linearity (R(2)≥0.9990), sensitivity (lower limit of quantitation of 0.05 ng mL(-1)), accuracy (83.3-108.3), precision (RSD≤15.6%) and stability (≥75.0%), and was approved for the determination OTA in plasma, heart, liver, spleen, lung, kidney and brain with a run time of 7.0 min. Simultaneously, an LC-TOF-MS method could unambiguously identify the metabolites of OTA in a total run time of 14 min. The subsequent studies on kinetics and distribution after oral administration of 0.2 mg/kg b.w. OTA in rat indicated that OTA could reach a maximum value of 1932.4±124.9 ng mL(-1) within 5h due to its fast absorption, and then was slowly eliminated in plasma with a half-life time (t1/2) of 75.6±29.0 h. Results of tissue accumulation after a daily oral administration of 0.1 mg/kg b.w. OTA during 20 days showed that the highest concentration of OTA was observed in lung (95.9±13.7 ng g(-1)), followed by liver (76.0±9.7 ng g(-1)), heart (62.0±4.2 ng g(-1)) and kidney (55.7±4.7 ng g(-1)). Furthermore, three less toxic metabolites of OTA were clearly identified: Ochratoxin β (OTβ) and ochratoxin B (OTB) methyl ester were found in kidney and spleen, respectively, while phenylalanine was detected in heart and kidney. Thus, a possible metabolic pathway of OTA was proposed. The above achieved results justified that the application of combinatorial LC-MS/MS and LC-TOF-MS methods are valuable tools to uncover the kinetics and metabolism of OTA for the interpretation of toxicological findings in animals and extrapolation of the resulting data as reference to humans.

A rabbit monoclonal antibody-based sensitive competitive indirect enzyme-linked immunoassay for rapid detection of chloramphenicol residue

A sensitive competitive indirect enzyme-linked immunoassay (ciELISA) based on a rabbit monoclonal antibody (RabMAb) against chloramphenicol (CAP) has been developed and validated in this study. After optimisation of several key physicochemical factors, such as Tween-20 percentage, pH value and ionic strength, the immunoassay showed excellent performance within the linear range of 0.18–6.37 ng mL−1, with the 50% inhibition concentration (IC50) of 1.06 ng mL−1 and the limit of detection (LOD) of 0.1 ng mL−1. In addition, the cross-reactivities of RabMAb towards chloramphenicol succinate, florfenicol and thiamphenicol were 2.09, 12.45 and 18.10%, respectively. Finally, the developed method was applied in spiked swine urine, milk and honey samples, with recoveries ranging from 71.03 to 109.62%. The result demonstrated that the developed immunoassay is a valuable method for screening and quantitation of CAP residues in real samples.

Multi-walled carbon nanotubes as solid-phase extraction sorbents for simultaneous determination of type A trichothecenes in maize, wheat and rice by ultra-high performance liquid chromatography-tandem mass spectrometry.

A solid-phase extraction (SPE) procedure using multi-walled carbon nanotubes (MWCNTs) as sorbents coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed for simultaneous determination of four type A trichothecenes in maize, wheat and rice for the first time. Several key parameters including the composition of sample loading solutions, washing and elution solvents were thoroughly investigated to achieve optimal SPE recoveries and efficiency. Performance of the MWCNTs materials was significantly affected by pH, and after optimization, n-hexane and 5% methanol aqueous solution as the washing solutions and methanol containing 1% formic acid as the elution solvent presented an excellent purification efficiency for the four targets in the different matrices. The method was validated by determining the linearity (R(2)≥0.992), recovery (73.4-113.7%), precision (1.2-17.1%) and sensitivity (limit of quantification in the range of 0.02-0.10μg/kg), and was further applied for simultaneous determination of type A trichothecenes in 30 samples. Although low contamination levels of type A trichothecenes in wheat, maize and rice were observed revealing mitigated risks to consumers in Shanghai, China, the developed method has proven to be a valuable tool for type A trichothecenes monitoring in complex crop matrices.

In Vitro Glucuronidation of Ochratoxin A by Rat Liver Microsomes

Ochratoxin A (OTA), one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), UHPLC-Orbitrap-high resolution mass spectrometry (HRMS) and liquid chromatography-multiple stage mass spectrometry (LC-MSn) were utilized to investigate the metabolic profile of OTA in rat liver microsomes. Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with β-glucuronidase. Moreover, OTA methyl ester, OTα and OTα-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time.