Zhaoying Liu

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.

Long-term mequindox treatment induced endocrine and reproductive toxicity via oxidative stress in male Wistar rats

Mequindox (MEQ) is a synthetic antimicrobial chemical of quinoxaline 1, 4-dioxide group. This study was designed to investigate the hypothesis that MEQ exerts testicular toxicity by causing oxidative stress and steroidal gene expression profiles and determine mechanism of MEQ testicular toxicity. In this study, adult male Wistar rats were fed with MEQ for 180days at five different doses as 0, 25, 55, 110 and 275mg/kg, respectively. In comparison to control, superoxide dismutase (SOD), reduced glutathione (GSH) and 8-hydroxydeoxyguanosine (8-OHdG) levels were elevated at 110 and 275mg/kg MEQ, whereas the malondialdehyde (MDA) level was slightly increase at only 275mg/kg. Furthermore, in LC/MS-IT-TOF analysis, one metabolite 2-isoethanol 4-desoxymequindox (M11) was found in the testis. There was significant decrease in body weight, testicular weight and testosterone at 275mg/kg, serum follicular stimulating hormone (FSH) at 110 and 275mg/kg, while lutinizing hormone (LH) levels were elevated at 110mg/kg. Moreover, histopathology of testis exhibited germ cell depletion, contraction of seminiferous tubules and disorganization of the tubular contents of testis. Compared with control, mRNA expression of StAR, P450scc and 17β-HSD in testis was significantly decreased after exposure of 275mg/kg MEQ while AR and 3β-HSD mRNA expression were significantly elevated at the 110mg/kg MEQ group. Taken together, our findings provide the first and direct evidence in vivo for the formation of free radicals during the MEQ metabolism through N→O group reduction, which may have implications to understand the possible mechanism of male infertility related to quinoxaline derivatives.

Metabolism of olaquindox in rat liver microsomes: structural elucidation of metabolites by high‐performance liquid chromatography combined with ion trap/time‐of‐flight mass spectrometry

Olaquindox (N-(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide) is a growth-promoting feed additive for food-producing animals. Its toxicity is closely related to the metabolism. The complete metabolic pathways of olaquindox are not revealed. To improve studies of the metabolism and toxicity of olaquindox, its biotransformation in rat liver microsomes and the structure of its metabolites using high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) were investigated. When olaquindox was incubated with an NADPH-generating system and rat liver microsomes, ten metabolites (M1-M10) were detected. The structures of these metabolites were identified from mass spectra and comparison of their changes in their accurate molecular masses and fragment ions with those of the parent drug. With the high resolution and good mass accuracy achieved by this technique, the elemental compositions of the metabolites and their fragment ions were exactly determined. The results indicate that the N --> O group reduction is the main metabolic pathway of olaquindox metabolism in rat liver microsomes, because abundant 1-desolaquindox (M2), 4-desolaquindox (M1) and bisdesoxyolaquindox (M9) were produced during the incubation step. Seven other minor metabolites were revealed which were considered to be hydroxylation metabolites, based on the position of the quinoxaline ring or 3-methyl group and a carboxylic acid derivative on the side chain at position 2 of the quinoxaline ring. Among the identified metabolites, five new hydroxylated metabolites (M3-M7) were found for the first time in rat liver microsomes. This work will conduce to complete clarification of olaquindox metabolism, and improve the in vivo metabolism of olaquindox in food animals.

Metabolism of cyadox in rat, chicken and pig liver microsomes and identification of metabolites by accurate mass measurements using electrospray ionization hybrid ion trap/time‐of‐flight mass spectrometry

Cyadox (CYX), (2-formylquinoxaline)-N(1),N(4)-dioxide cyanoacetylhydrazone, is a growth promoter, which is more efficient and less toxic to animals. Few studies have been performed to reveal the metabolism of CYX in animals till now. In this study, the metabolic fate of CYX in the liver microsomes of animal was investigated firstly using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. CYX was incubated with rat, chicken and pig liver microsomes in the presence of a NADPH-generating system. Multiple scans of metabolites in MS and MS(2) modes and accurate mass measurements were performed simultaneously through data-dependent acquisition. Most measured mass errors were less than 10 ppm for both protonated molecules and fragment ions using external mass calibration. The structures of metabolites and their fragment ions were easily and reliably characterized based on the accurate MS(2) spectra and known structure of CYX. The relative biotransformation of CYX into characterized metabolites was estimated based on the UV absorption and the assumption that all metabolites had the same extinction coefficient as the parent compound at 305 nm. Totally, seven metabolites were identified as three reduced metabolites (cyadox 1-monoxide (Cy1), cyadox 4-monoxide (Cy2) and bisdesoxycyadox (Cy4)), three hydrolysis metabolites of the amide bond (N-decyanoacetyl cyadox (Cy5), N-decyanoacetyl cyadox 1-monoxide (Cy6) and N-decyanoacetyl bisdesoxycyadox (Cy7)) and a hydroxylation metabolite of Cy1 (Cy3). Cy1-Cy6 could be detected in rat, chicken and pig liver microsomes while metabolite Cy7 could only be observed in pig. The amounts of the metabolites in three species are different. For the formations of Cy1 and Cy3, the rank order was rat approximately chicken > pig. For Cy4 and Cy5, the order was pig > rat > chicken. Cy1 and Cy4 have been previously reported, whereas the other five metabolites were novel. The N-->O group reduction and hydroxylation were the main metabolic pathways for CYX in the three species.

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.

Metabolites and JAK/STAT pathway were involved in the liver and spleen damage in male Wistar rats fed with mequindox

Mequindox (MEQ) is a novel synthetic quinoxaline 1,4-dioxides antibacterial agent and growth promoter in animal husbandry. This study was to investigate whether reactive oxygen species (ROS), the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway, suppressors of cytokine signaling (SOCS) and inflammatory cytokines were involved in toxicities of MEQ. Our data demonstrated that high dose of MEQ (275 mg/kg) apparently led to tissue impairment combined with imbalance of redox in liver. In liver and spleen samples, hydroxylation metabolites and desoxymequindox were detected, directly confirming the potential link of N→O group reduction metabolism with its organ toxicity. Moreover, up-regulation of JAK/STAT, SOCS family, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) were also observed in the high-dose group. Meanwhile, significant changes of oxidative stress indices in liver were observed in the high-dose group. As for NADPH subunit, the mRNA levels of many subunits were significantly up-regulated at low doses but down-regulated in a dose-dependent manner in liver and spleen, suggesting an involvement of NADPH in MEQ metabolism and ROS generation. In conclusion, we reported the dose-dependent long-term toxicity as well as the discussion of the potential mechanism and pathways of MEQ, which raised further awareness of its toxicity following with the dose change.

Metabolism of mequindox in liver microsomes of rats, chicken and pigs

Mequindox, 3-methyl-2-quinoxalinacetyl-1,4-dioxide, is a quinoxaline-N,N-dioxide used in veterinary medicine as a antibacterial in China. To gain an understanding of the interspecies differences in the metabolism of mequindox, comparative metabolite profiles were qualitatively and quantitatively carried out for the first time in rat, chicken and pig liver microsomes by high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. A total of 14 metabolites were characterized based on their accurate MS(2) spectra and known structure of mequindox. The in vitro metabolic pathways of mequindox in three species were proposed as N-->O group reduction, carbonyl reduction, N-->O group reduction followed by carbonyl reduction or methyl mono-hydroxylation. A metabolic pathway involving N-->O group reduction followed by acetyl group mono-hydroxylation in only chicken was also proposed. There was also quantitative species difference for mequindox metabolism in three species. 1-Desoxymequindox was the main metabolite in all species, but otherwise there were some qualitative interspecies differences in mequindox major metabolites. This work has revealed biotransformation characteristics of mequindox among different species, and moreover will further facilitate the explanations of the biological activities of mequindox in animals.

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.

The metabolism and N-oxide reduction of olaquindox in liver preparations of rats, pigs and chicken.

Olaquindox, N-(2-hydroxyethyl)-3-methyl-2-quinoxalinecarboxamide-1,4-di-N-oxide, is one of the quinoxaline-dioxides used widely as an antimicrobial growth promoter in pig production. Its toxicities were reported to be closely related to the formation of N-oxide reductive metabolites. The present study presents the metabolism and N-oxide reduction of olaquindox incubated with liver microsomes and liver cytosol of rats, pigs and chicken. Metabolites were identified and characterized with a novel LC/MS-ITTOF. Thirteen metabolites were found in liver microsomes of rats, three of which were identified to be novel. Seven metabolites were found in liver microsomes of pigs and chicken. The N-oxide reduction was the major metabolic pathway of olaquindox in liver microsomes of the three species. The N1-reduction of olaquindox to metabolite O2 was found in not only liver microsomes but also cytosol of the three species in the presence of NAD(P)H under hypoxic conditions. The N1-reduction could be inhibited by air and carbon monoxide, and be significantly stimulated by riboflavin under various conditions. The N1-reduction in the liver cytosol of rats and pigs could be enhanced by menadione, but the reduction in liver cytosol of chicken could not be. The N1-reduction activities in all animals were not abolished when liver microsomes and cytosol were boiled. These findings suggested that the N1-reduction of olaquindox could be mediated by non-enzymatic and enzymatic conditions. This N1-reduction of olaquindox could also be catalyzed by a quinone-dependent reducing system in liver cytosol of rats and pigs. Moreover, liver cytosol of rats and pigs had an ability of N4-reduction that catalyzed olaquindox to metabolite O1 in the presence of benzaldehyde under hypoxic conditions, but the liver cytosol of chicken did not. The N4-reduction could be inhibited markedly in the cytosol rats and pigs by menadione, chlorpromazine and promethazine. In addition, 7-hydroxycoumarin was also found to inhibit the formation of O1 in the cytosol of rats. The inhibitory results suggested that the N4-reduction might be catalyzed by aldehyde oxidase in the cytosol of pigs, and by aldehyde oxidase and xanthine oxidase in the cytosol of rats. In conclusion, the N1-reduction and N4-reduction of olaquindox are mediated by multiple mechanisms and significant species differences are involved in both reductions. This work is a contribution to the understanding of toxicities and the relativities between toxicities and metabolism of olaquindox.

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

A multi-residues method using pressurized liquid extraction (PLE) and liquid chromatography combined with mass spectrometry (LC-MS/MS) has been developed for determination of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, fludrocortisone) in muscle of swine, cattle, and sheep. Parameters affecting PLE extraction including extraction solvent, extraction temperature, extraction pressure and extraction cycles were optimized. The optimized method employed 11 ml extraction cells, hexane-ethyl acetate (50:50, v/v) as extraction solvent, 1500 psi of extraction pressure and 50°C of extraction temperature. The samples were detected by LC-ESI-MS/MS in negative mode with selected reaction monitoring (SRM) mode. The recovery of glucocorticoids spiked at levels of 0.5-6 μg kg(-1) ranged from 70.1% to 103.1%; the between-day relative standard deviations were no more than 9.6%. The limits of quantification were 0.5-2 μg kg(-1) in muscle. The results demonstrated that the method is simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.