Yuanhu Pan

Simultaneous determination of 15 aminoglycoside(s) residues in animal derived foods by automated solid-phase extraction and liquid chromatography-tandem mass spectrometry.

An automated method has been developed for the simultaneous quantification of 15 aminoglycosides in muscle, liver (pigs, chicken and cattle), kidney (pigs and cattle), cow milk, and hen eggs by liquid chromatography tandem mass spectrometry. Homogenized samples were extracted by monopotassium phosphate buffer (including ethylene diamine tetraacetic acid), and cleaned up with auto solid-phase extraction by carboxylic acid cartridges. The analytes were separated by a specialized column for aminoglycosides, and eluted with trifluoroacetic acid and acetonitrile. The decision limits (CCα) of apramycin, gentamycin, tobramycin, paromomycin, hygromycin, neomycin, kanamycin, sisomicin, netilmicin, ribostamycin, kasugamycin, amikacin, streptomycin, dihydrostreptomycin and spectinomycin were ranged from 8.1 to 11.8 μg/kg and detection capabilities (CCβ) from 16.4 to 21.8 μg/kg. High correlation coefficients (r(2)>0.99) of calibration curves for the analytes were obtained within linear from 20 to 1000 μg/kg. Reasonable recoveries (71-108%) were demonstrated with excellent relative standard deviation (RSD). This method is simple pretreatment, rapid determination and high sensitivity, which can be used in the determination of multi-aminoglycosides in complex samples.

Genotoxicity of quinocetone, cyadox and olaquindox in vitro and in vivo

Quinocetone (QCT) and Cyadox (CYA) are important derivative of heterocyclic N-oxide quinoxaline (QdNO), used actively as antimicrobial feed additives in China. Here, we tested and compared the genotoxic potential of QCT and CYA with olaquindox (OLA) in Ames test, HGPRT gene mutation (HGM) test in V79 cells, unscheduled DNA synthesis (UDS) assay in human peripheral lymphocytes, chromosome aberration (CA) test, and micronucleus (MN) test in mice bone marrow. OLA was found genotoxic in all 5 assays. In Ames test, QCT produced His(+) mutants at 6.9 μg/plate in Salmonella typhimurium TA 97, at 18.2 μg/plate in TA 100, TA 1535, TA 1537, and at 50 μg/plate in TA 98. CYA produced His(+) mutants at 18.2 μg/plate in TA 97, TA 1535, and at 50 μg/plate in TA 98, TA 100 and TA 1537. QCT was found positive in HGM and UDS assay at concentrations ≥10 μg/ml while negative results were reported in CA test and MN test. Collectively, we found that OLA was more genotoxic than QCT and CYA. Genotoxicity of QCT was found at higher concentration levels in Ames test, HGM and UDS assays while CYA showed weak mutagenic potential to bacterial cells in Ames test.

Development of a high performance liquid chromatography method and a liquid chromatography―tandem mass spectrometry method with pressurized liquid extraction for simultaneous quantification and confirmation of cyromazine, melamine and its metabolites in foods of animal origin

Simple and sensitive methods have been developed for simultaneous detection of cyromazine, melamine and their metabolites (ammeline, ammelide and cyanuric acid) in samples of animal origins. These include a high performance liquid chromatography (HPLC) method and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and are useful in regular monitoring and in toxicity studies of these molecules. Representative samples used in this study include muscles and livers of swine, bovine, sheep and chicken, kidneys of swine, bovine and sheep, and milk powder. A new sample preparation procedure with pressurized liquid extraction (PLE) at 1400psi and 70°C was investigated. Quantification of these five compounds by HPLC was achieved using an APS-2 column with UV detection at 230 nm. Limit of detection (LOD) was at 10 μgkg(-1), and limit of quantification (LOQ) was at 40 μgkg(-1). Recoveries of the five analytes in spiked samples ranged from 72.2% to 115.4% with RSD less than 12%. Confirmatory analysis of the analytes was performed using LC-MS/MS in selected reaction monitoring (SRM) mode. The LOD and LOQ were 5 μgkg(-1) and 15 μgkg(-1), respectively. This is the first simultaneous analysis of cyromazine, melamine, ammeline, ammelide and cyanuric acid residues in complex tissue samples using PLE and HPLC. It is expected that these methods will find many practical applications in evaluating the safety of cyromazine, melamine and their metabolites.

Development of a sensitive and robust liquid chromatography coupled with tandem mass spectrometry and a pressurized liquid extraction for the determination of aflatoxins and ochratoxin A in animal derived foods

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a pressurized liquid extraction (PLE) was developed for the simultaneous determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and M₂, and ochratoxin A in the muscle, liver, kidney and fat of swine, bovine and sheep, muscle and liver of chicken, muscle and skin of fish, as well as in hen eggs and dairy milk. Samples were extracted with PLE and cleaned-up with solid phase extraction (SPE) on HLB cartridges. The optimum extraction conditions were obtained as a 11 ml ASE cell, acetonitrile/hexane as the extraction solvent, 1500 psi, 100 °C, a 5 min static time and a 60% flush volume. A cheaper and widely used SPE column (Oasis HLB) was applied during clean up. The detection and quantification of the 7 mycotoxins were performed by a reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS). The limits of detection defined as CCα varied from 0.07 μg/kg to 0.59 μg/kg. The recoveries of spiked samples from 0.25 μg/kg to 1 μg/kg ranged from 68.3% to 105.7% with the relative standard deviations of less than 17.6%. Performances of the whole analytical procedure met the criteria established by the European Commission for mass spectrometric detection.

Biodegradable nanoparticles for intracellular delivery of antimicrobial agents

Biodegradable nanoparticles have emerged as a promising strategy for ferrying antimicrobial agents into specific cells due to their unique properties. This review discusses the current progress and challenges of biodegradable nanoparticles for intracellular antimicrobial delivery to understand design principles for the development of ideal nanocarriers. The intracellular delivery performances of biodegradable nanoparticles for diverse antimicrobial agents are first summarized. Second, the cellular internalization and intracellular trafficking, degradation and release kinetics of nanoparticles as well as their relation with intracellular delivery of encapsulated antimicrobial agents are provided. Third, the influences of nanoparticle properties on the cellular internalization and intracellular fate of nanoparticles and their payload antimicrobial agents are discussed. Finally, the challenges and perspectives of nanoparticles for intracellular delivery of antimicrobial agents are addressed. The review will be helpful to the scientists who are interested in searching for more efficient nanosystem strategies for intracellular delivery of antimicrobial agents.

Development of a liquid chromatography-tandem mass spectrometry with pressurized liquid extraction method for the determination of benzimidazole residues in edible tissues

A confirmatory and quantitative method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with a pressure liquid extraction (PLE) was developed for the determination of 11 benzimidazole and 10 metabolites of albendazole, fenbendazole and mebendazole in the muscles and livers of swine, cattle, sheep and chicken. For sample preparation, we used an automated technique of PLE method. The optimum extraction conditions were obtained using an 11 ml Accelerated Solvent Extraction (ASE) cells, acetonitrile/hexane as the extraction solvent. HPLC analysis was performed on a C18 column with gradient elution using acetonitrile and 5 mmol l(-1) formic ammonium as mobile phase. The analytes were detected in the positive ion multiple reaction monitoring (MRM) mode by the LC-ESI-MS/MS analysis. The recoveries of benzimidazole (BZDs) spiked at the levels of 0.5 μg kg(-1) ranged from 70.1% to 92.7%; the between-day relative standard deviations were no more than 10%. The limits of quantification were 0.02-0.5 μg kg(-1). The optimized method was successfully applied to monitor real samples containing BZDs, demonstrating the method to be simple, fast, robust and suitable for identification and quantification of BZDs residues in animal products.

Development of a high-performance liquid chromatography method to monitor the residues of benzimidazoles in bovine milk.

A reversed-phase high-performance liquid chromatography with ultraviolet (UV) detection was developed that can determine 11 benzimidazole (BZDs) and 10 metabolites of albendazole, fenbendazole and mebendazole in bovine milk. Samples were extracted with acetonitrile and purified by mixed-mode cation exchange (MCX) solid phase extraction cartridges. LC separations were performed on a C(18) column with gradient elution using acetonitrile and ammonium acetate solution. The UV-detection was set at 292nm. The method is very sensitive to each analyte with limits of quantification (LOQs) of lower than 10μgkg(-1). The recoveries of the BZDs and their metabolites spiked in milk were more than 78% with between-day relative standard deviation values less than 16% at the concentration of 10, 50 and 100μgkg(-1). The method developed has been successfully applied to monitoring real samples containing BZDs, which demonstrated that it is a simple, fast and robust method, and could be used as a regulatory toll to determine the residues of BZDs in milk.

Deoxidation rates play a critical role in DNA damage mediated by important synthetic drugs, quinoxaline 1,4-dioxides.

Quinoxaline 1,4-dioxides (QdNOs) are synthetic agents with a wide range of biological activities. However, the mechanism of DNA damage mediated by QdNOs is far from clear. Five classical QdNOs, quinocetone (QCT), mequindox (MEQ), carbadox (CBX), olaquindox (OLA), and cyadox (CYA), were used to investigate the genotoxicity of QdNOs. The deoxidation rate of QdNOs was presumed to play a role in their genotoxicity. Deoxidation rates of QdNOs in both rat and pig liver microsomes were investigated using LC/MS-IT/TOF, and their relative quantification was achieved with HPLC. To reveal the relationships between the deoxidation rate and genotoxicity, cell damage, oxidative stress, and DNA damage were detected. Under low oxygen conditions, the rank order of the desoxy and bidesoxy rates in rat and pig liver microsomes was QCT < CBX < MEQ < OLA < CYA and QCT < MEQ < CBX < OLA < CYA, respectively. Only desoxy-quinoxalines were detected under aerobic conditions. The concentrations of deoxidized metabolites under low oxygen conditions were at least 6 times higher than those under aerobic conditions. In rats, porcine primary hepatocytes, and HepG2 cells, oxidative stress indices and DNA damage showed inverse relationships with the deoxidation rate, indicating that the deoxidation rate of QdNOs, especially bidesoxy rates, might play a critical role in mediating their ability to promote DNA damage. These results indicated that faster deoxidation of QdNOs results in lower DNA-damage-induced toxicity. Our results shed new light on the prevention of DNA damage mediated by QdNOs and help to understand the relationships among the chemical structures, metabolism, and DNA damage of QdNOs.

Development of a liquid chromatography–tandem mass spectrometry with ultrasound-assisted extraction method for the simultaneous determination of sudan dyes and their metabolites in the edible tissues and eggs of food-producing animals

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of sudan I, sudan II, sudan III, sudan IV, and their metabolites such as 4-aminoazobenzene and ortho-aminoazotoluole in 12 animal derived foods (including the muscle and liver of swine, muscle, liver and skin of chicken and duck, muscle and skin of fish, as well as the eggs of hen and duck). Sample preparation procedure included ultrasound-assisted extraction with acetonitrile, defatting with n-hexane and final clean-up with solid phase extraction (SPE) on Aluminum B cartridges. The detection and quantification of the 6 sudan dyes and their metabolites were performed by a reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS). The CCαs and the CCβs of various samples varied from 0.03μg/kg to 0.12μg/kg, 0.09μg/kg to 0.19μg/kg, respectively. The recoveries of spiked sample from 0.2μg/kg to 0.8μg/kg ranged from 61.9% to 87.4% with the relative standard deviations of less than 19.1%. Performances of the whole analytical procedure meet the criteria established by the European Commission for mass spectrometric detection.

Metabolism, distribution, and excretion of deoxynivalenol with combined techniques of radiotracing, high-performance liquid chromatography ion trap time-of-flight mass spectrometry, and online radiometric detection.

Dispositions of deoxynivalenol (DON) in rats and chickens were investigated, using a radiotracer method coupled with a novel γ-accurate radioisotope counting (γ-ARC) radio-high-performance liquid chromatography ion trap time-of-flight tandem mass spectrometry (radio-HPLC-IT-TOF-MS/MS) system. 3β-(3)H-DON was chemically synthesized and orally administrated to both sexes of rats and chickens as single or multiple doses. The results showed that DON was widely distributed and quickly eliminated in all tissues. The highest concentration was found in the gastrointestinal tract at 6 h post-administration. Substantially lower levels were detected in the kidney, liver, heart, lung, spleen, and brain. Three new metabolites were identified tentatively as 10-deoxynivalenol-sulfonate, 10-deepoxy-deoxynivalenol (DOM-1)-sulfonate, and deoxynivalenol-3α-sulfate. Deoxynivalenol-3α-sulfate was a major metabolite in chickens, while the major forms in rats were DOM-1 and DON. Additionally, a higher excretion rate in urine was observed in female rats than in male rats. The differences in metabolite profiles and excretion rates, which suggested diverse ways to detoxify, may relate to the different tolerances in different genders or species.