Dapeng Peng

Aqueous two-phase system (ATPS): an overview and advances in its applications

Aqueous two-phase system (ATPS) is a liquid-liquid fractionation technique and has gained an interest because of great potential for the extraction, separation, purification and enrichment of proteins, membranes, viruses, enzymes, nucleic acids and other biomolecules both in industry and academia. Although, the partition behavior involved in the method is complex and difficult to predict. Current research shows that it has also been successfully used in the detection of veterinary drug residues in food, separation of precious metals, sewage treatment and a variety of other purposes. The ATPS is able to give high recovery yield and is easily to scale up. It is also very economic and environment friendly method. The aim of this review is to overview the basics of ATPS, optimization and its applications.

Acute and sub-chronic oral toxicological evaluations of quinocetone in Wistar rats

To provide a detailed toxicity with wide spectrum of doses for quinocetone, a new antimicrobial growth promoting agent, acute and sub-chronic toxicological studies were conducted. For acute study, quinocetone was administered singly by oral gavage to Wistar rats and Kunming mice. Calculated LD50 was 8687.31 mg/kg b.w./day in rats and 15848.93 mg/kg b.w./day in mice. In sub-chronic study, quinocetone was fed to Wistar rats at dietary levels of 0, 50, 300 and 1800 mg/kg or olaquindox (300 mg/kg), approximately equivalent to quinocetone 5, 30, 180 or olaquindox 30 mg/kg b.w./day. There was significant decrease in body weight in both genders, total protein and creatinine in females and alkaline phosphatase in males fed with 1800 mg/kg diet, while alkaline aminotransferase values decreased in all treated groups. Significant increase in relative weights of liver and kidneys in both genders and testis in male rats were noted at 1800 mg/kg diet. Histopathological observations revealed that 1800 mg/kg quinocetone diet and 300 mg/kg olaquindox diet could induce proliferation of bile canaliculi in the portal area. In conclusion, quinocetone can induce hepatic histological changes as well as leaking of different serum enzymes. The no-observed-adverse-effect level of quinocetone was considered to be 300 mg/kg diet.

Development of a high performance liquid chromatography method and a liquid chromatography–tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulfonamides in the foods of animal origin

The residues of sulfonamides (SAs) in the foods of animal origin are of the major concern because they are harmful to the consumers health and could induce pathogens to develop resistance. Rapid and efficient determination methods are urgently in need. A quantitative high performance liquid chromatography method (HPLC) and a confirmative liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of 18 sulfonamides such as sulfamidinum, sulfanilamide, sulfisomidine, sulfadiazine, sulfapyridine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamethoxypyridazine, sulfamethoxydiazine, sulfisoxazole, sulfachloropyridazine, sulfamethoxazole, sulfamonomethoxine, sulfadoxine, sulfaclozine, sulfadimethoxine, sulfaquinoxaline in the muscles, livers and kidneys of swine, bovine and chicken were developed and validated. The sample preparation procedures included a pressurized liquid extraction (PLE) with acetonitrile conducted at elevated temperature (70°C) and pressure (1400 psi). After clean-up with hydrophilic-lipophilic balance cartridge, the extraction solution was concentrated and analyzed by HPLC and LC-MS/MS analysis. 18 SAs were separated by the HPLC with a Zorbax SB-Aq-C18 column and the mobile phase of methanol/acetonitrile/1% acetic acid with a gradient system. The wavelength of UV for the HPLC detection was set at 285 nm. The LC-MS/MS analysis was achieved with a Hypersil Golden column and the mobile phase of acetonitrile and 0.1% formic acid aqueous solution with two gradient systems. The Limits of detection (LOD) and the limits of quantitation (LOQ) were 3 μg/kg and 10 μg/kg, respectively, for both of the HPLC and LC-MS/MS. Linearity was obtained with an average coefficient of determination (R) higher than 0.9980 over a dynamic range from the LOQ value up to 5000 μg/kg. The recoveries of the methods range from 71.1% to 118.3% with the relative standard derivation less than 13%. The peaks of interest with no interferences were observed throughout the chromatographic run. The sample pretreatment provided efficient extraction and cleanup that enables a sensitive and rugged determination of 18 SAs, the obtained results revealed that PLE, in comparison with other sample preparation methods applied, has significantly higher efficacy for SAs isolation from animal tissues.

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.

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.

23S rRNA mutation A2074C conferring high-level macrolide resistance and fitness cost in Campylobacter jejuni.

To examine the development of macrolide resistance in Campylobacter jejuni and assess the fitness of the macrolide-resistant mutants, two macrolide-susceptible C. jejuni strains, American Type Culture Collection (ATCC) 33291 and H1, from different geographic areas were exposed to tylosin in vitro. Multiple mutant strains were obtained from the selection. Most of the high-level macrolide-resistant strains derived from the selection exhibited the A2074C transversion in all three copies of 23S rRNA and displayed strong stability in the absence of antibiotic selection pressure. The competition experiments demonstrated that the strains containing the A2074C transversion imposed a fitness cost in competition mixtures. In addition, the fitness cost of the mutation was not ameliorated after approximately 500 generations of evolution under laboratory conditions. These findings indicate that the A2074C transversion in C. jejuni is not only correlated with stable and high-level macrolide resistance but also associated with a fitness cost.

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.

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.

Simultaneous determination of fluoroquinolones in foods of animal origin by a high performance liquid chromatography and a liquid chromatography tandem mass spectrometry with accelerated solvent extraction

A confirmatory and quantitative method based on a high performance liquid chromatography UV detector (HPLC-UV) and a liquid chromatography tandem mass spectrometry (LC-MS/MS) with an extraction procedure of accelerated solvent extraction (ASE) has been developed for simultaneous determination of 15 kinds of fluoroquinolones in various animal origin food samples. The sample preparation procedures consist of an extraction step with acetonitrile and a cleaning-up step with Oasis HLB cartridge. Parameters for extraction pressure and temperature, cycle of ASE, clean-up, and analysis procedure have been optimized systematically. The recoveries of FQNs spiked in the tissues as the muscle, liver, kidney of swine, bovine, chicken and fish at a concentration range of 10-800μg/kg were found between 70.6% and 111.1% with relative standard deviations (RSD) less than 15% in HPLC. The LOD and LOQ of the HPLC for the 15 FQNs were 3μg/kg and 10μg/kg, respectively, and those of the LC-MS/MS were 0.3 and 1μg/kg, respectively. These rapid and reliable methods can be used to efficiently separate, characterize and quantify the residues of 15 FQNs (Marbofloxacin, Enoxacin, Fleroxacin, Ofloxacin, Pefloxacin, Lomefloxacin, Danofloxacin, Enrofloxacin, Orbifloxacin, Cinoxacin, Gatifloxacin, Sarafloxacin, Difloxacin, Nalidixic Acid, Flumequine) in food of animal origin.