Residues In Animal Tissues Research Articles

Rapid untargeted screening for drug residues in animal tissues with liquid microjunction surface sampling probe mass spectrometry

An untargeted screening method for the rapid identification of veterinary drug residues in incurred animal tissues using liquid microjunction surface sampling probe mass spectrometry (LMJSSP-MS) was developed. Current analytical methods for veterinary drug residue screening involve lengthy sample preparation, extraction, and instrumental analysis steps. This method identifies veterinary drug residues in several different incurred animal tissues more quickly than conventional analytical methods. This LMJSSP-MS method uses an ambient ionization technology called liquid microjunction surface sampling probe along with a data dependent scan function of a quadrupole orbitrap mass spectrometer. Collected product ion spectra are searched against the mzCloud™ online mass spectral database to identify veterinary drug residues found in incurred animal tissue samples. Examples of veterinary drugs identified with this method include flunixin, tilmicosin, pentobarbital, xylazine, and ketamine. Optimization of method parameters is described and discussed. The limit of identification (LOI) of this method is estimated to be approximately 1 μg g−1 for xylazine and ketamine.

Determination of tylosin residues in animal tissues by HPLCPDA.

Tylosin belongs to the group of macrolide antibiotics and is widely used both therapeutically against Gram-positive microorganisms and Mycoplasma spp. and as a feed additive for growth promotion. In the European Union, according to Regulation 1442/95, tylosin residues of up to 100 μg/kg are permitted in the edible tissues of slaughter animals. For the determination of tylosin residues in meat and kidney samples a modification of the method of Chan et al. (1994) has been applied. The method is simple, rapid and accurate. Tylosin extraction is carried out with acetonitrile combined with phosphate buffer (pH 2.5) and extract eleanup is performed using BondElut C18 s.p. cartridges. Tylosin is luted with 0.1 M ammonium acetate in methanol. The determination is accomplished by RP-HPLC with UV detection at 287 nm. The LC separation is carried out on a Symmetry C18 column (150x3.9 mm, 5μ) using a mobile phase of 0.1M ammonium formate (pH 5.0) / acetonitrile (70/30) at a flow rate of 0.8 ml/min. Positive results are easily confirmed by spectra comparison. The recovery achieved was 86.70±0.95% and the limit of determination 40 μg/kg. Diluted standards and sample extracts are stable for 3 weeks if kept at -20 °C.

Immunochromatographic Assay for Simultaneous and Quantitative Detection of 3-Methyl-Quinoxaline-2-Carboxylic Acid and Quinoxaline-2-Carboxylic Acid Residues in Animal Tissues Based on Highly Luminescent Quantum Dot Beads

Due to their potential adverse effects on human health, the use of carbadox and olaquindox in feedingstuffs was prohibited by the European Union since 1998. In this work, highly luminescent quantum dot beads (QBs) were synthesized by encapsulating CdSe/ZnS and used as novel fluorescent probes in the immunochromatographic assay (ICA) for simultaneous and quantitative determination of metabolites of olaquindox (3-methylquinoxaline-2-carboxylic acid, MQCA) and carbadox (quinoxaline-2-carboxylic, QCA). The fluorescence intensities of the test lines were recorded using a fluorescence strip reader. The 50% of inhibition for MQCA and QCA was shown to be 8.1 and 10.6 μg L−1, respectively. The whole assay process can be accomplished within 10 min. The immunosensor was used to analyze spiked samples, and analyte intra- and inter-assay recovery rates ranged from 78.7 to 92.2% for MQCA and 80.6 to 95.8% for QCA, and coefficients of variation were all below 12%. The incurred tissues samples were assayed using both QB-based ICA and commercial ELISA kit and were further confirmed with LC-MS/MS. The QB-based ICA results exhibited good agreement with both commercial ELISA and LC-MS/MS methods.

β-cyclodextrin-ionic liquid polymer based dynamically coating for simultaneous determination of tetracyclines by capillary electrophoresis.

Tetracyclines are a group of broad spectrum antibiotics widely used in animal husbandry to prevent and treat diseases. However, the improper use of tetracyclines may result in the presence of their residues in animal tissues or waste. Recently, great attention has been drawn towards the green solvents ionic liquids. Ionic liquids have been employed as a coating material to modify the electroosmotic flow in capillary electrophoresis. In this study, a functionalized ionic liquid, mono-6-deoxy-6-(3-methylimidazolium)-β-cyclodextrin tosylate, was synthesized and used for the simultaneous separation and quantification of tetracyclines by capillary electrophoresis. Good separation efficiency could be achieved due to the multiple functions of β-cyclodextrin derived ionic liquid, including the electrostatic interaction, the hydrogen bonding, and the cavity structure in β-cyclodextrin ionic liquid which can entrap the tetracyclines to form inclusion complex. After optimization, baseline separation achieved in 25 min with the running buffer consisted of 10 mmol/L, pH 7.2 phosphate buffer and 20 mmol/L β-cyclodextrin ionic liquid. The satisfied result demonstrated that the β-cyclodextrin ionic liquid is an ideal background electrolyte modifier in the separation of tetracyclines with high stability and good reproducibility. And it is an effective strategy to design and synthesize specific ILs as additive applied in separation.

Dual-label quantum dot-based immunoassay for simultaneous determination of Carbadox and Olaquindox metabolites in animal tissues

A novel and reliable dual-label direct competitive fluorescence-linked immunosorbent assay (dc-FLISA) based on quantum dots (QDs) was developed for the simultaneous determination of the major metabolites of Carbadox and Olaquindox residues in animal tissues, using anti-QCA monoclonal antibodies and anti-MQCA polyclonal antibodies labeled with QD520 and QD635, respectively. The limits of detection for QCA and MQCA were 0.05 and 0.07ng/ml, respectively. The method was used to analyze fortified samples and analyte recoveries ranged from 81.5% to 98.2% (QCA) and 84.2% to 95.7% (MQCA). Good correlations between the dc-FLISA method and HPLC were demonstrated for the determination of QCA and MQCA residues in swine tissue samples, confirming the reliability of the proposed method.

QuEChERS 법을 이용한 Rat 조직내 Pyraclofos 잔류 분석 및 급성독성 평가

Abstract Environmental pesticides used for insect control can be transferred from plants to animals even to live-stock animals through food chain. Human beings also can be exposed to pesticides by consuming pol-luted dairy products, including meats, eggs and other milk products. Therefore, the Ministry of Food and Drug Safety (MFDS) established Standard for Pesticide Residue Limits in dairy products. The QuEChERS (quick, easy, cheap, effective, rugged and safe) methods for detecting residual pesticides are relatively well established for fruits and vegetables, however, the methods for meat have not been appropriately studied yet. In the present work, pyraclofos was used as an organophosphate pesticide to examine its tissue residue in experimental animals by QuEChERS methods. For this, pyraclofos (150 mg/kg body weight) was orally administered to male rats once a day for 2 days. After 6, 12, and 24 hr of the treatment, the tissue residues in liver and femoral muscle of the rats were determined using QuEChERS methods followed by HPLC analyses. In preliminary studies, the recovery rates of spiking samples of pyraclofos demonstrated approximately 109∼110% from the tissues. In previous study, pyr-aclofos tissue residues were observed with significantly high levels in livers and muscles at 6 hr of oral treatment. Then, they were almost completely disappeared after 24 hr of the administration, indicating the orally exposed pyraclofos is rapidly absorbed and distributed to body organs, then quickly excreted from the body with a negligible level of tissue residue. The alterations in blood chemistry as well as the histopathology of heart, lung, liver, spleen and kidney have also been investigated in the ex-perimental animals for assessing acute toxic effects of pyraclofos. The obtained blood chemistry indexes (ALT and AST) showed maximum peak values at 12 hr after the oral administration and decreased to the normal levels at 24 hr of the treatment. Histopathologic observation exhibited acute hepatic damages at 24 hr of the treatment. In conclusion, we suggest that QuEChERS method can be adequately opti-mized for the analysis of pyraclofos residues in animal tissues.

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for the detection of albendazole 2-aminosulfone residues in animal tissues

ABSTRACTTo monitor the residues of albendazole (ABZ) in edible animal tissues, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed. The hapten albendazole 2-aminosulfone (ABZSO2NH2) was conjugated to a carrier protein as an immunogen. The spleen cells of the inoculated mice expressing specificity to ABZSO2NH2 were fused. The obtained monoclonal antibody 2A11 was selected for detailed study. The standard curves based on the ABZSO2NH2 matrix calibration ranged from 20.0 to 320.0 µg L−1, with an IC50 value of 85.2 ± 6.3 µg L−1 (n = 5). The limits of detection for ABZSO2NH2 ranged from 11.0 to 20.8 µg kg−1 in edible animal tissues. The recoveries ranged from 73.6% to 99.8% with a coefficient of variation of less than 20%. The developed ic-ELISA exhibited good correlation (r = 0.999) with high-performance liquid chromatography. Thus, it would be a very attractive alternative for the screening of ABZSO2NH2 residues in edible animal tissues.

Simultaneous Determination of Malachite Green, Diethylstilbestrol, Medroxyprogesterone, and 3-Amino-2-Oxazolidone in Synbranchoid Eels by a Protein Microarray Method

Protein chips have emerged as a useful approach for a wide variety of applications, including the identification of small molecule targets. In this study, a protein microarray method was developed for the simultaneous detection of the following four veterinary drug residues in synbranchoid eels: leucomalachite green (LMG), diethylstilbestrol (DES), medroxyprogesterone (MPA), and 3-amino-2-oxazolidone (AOZ). The analytes were coupled to ovalbumin (OVA) before immobilization on the surface of modified glass slides. After immobilization, a mixture of antibodies to the four analytes and either standard solutions containing the analytes or samples were added to the array reaction area. Then, Cy3-labeled goat anti-rabbit IgG was added and analyte residues were detected quantitatively. The results indicated that the 50 % inhibitory concentrations of DES, MPA, LMG, and AOZ were 2.0, 1.98, 1.65, and 0.355 μg/L, respectively. The limit of detection of DES, MPA, LMG, and AOZ in synbranchoid eels were 0.325, 0.37, 0.271, and 0.103 μg/kg, respectively, and recovery rates ranged from 60.7 to 98.6 % for fortified samples at levels of 2, 5, and 10 μg/kg with coefficient of variation values <15 %. This protein microarray method was compared with commercially available kits for these four analytes; both methods yielded similar results. These results indicate that the protein microarray method is suitable for the detection of DES, MPA, LMG, and AOZ, as well as other potential drug residues in animal tissues.

Development of a Rapid Method for the Confirmatory Analysis of Flunixin Residue in Animal Tissues Using Liquid Chromatography–Tandem Mass Spectrometry

Flunixin is approved for use in veterinary medicine as a nonsteroidal anti-inflammatory agent. There is a need for the control of residues and development of methods to monitor its compliance with legislation. However, few methods have been reported for the analysis of flunixin in the animal liver, kidney, and fat. Therefore, the aim of this work was to develop a relatively rapid sample preparation and sensitive using liquid chromatography–tandem mass (LC-MS/MS) confirmatory method to detect flunixin residues in the liver, kidney, muscle, and fat of swine and chicken. After acid hydrolysis, the sample was extracted with ethyl acetate. The extract was finally evaporated to dryness and reconstituted in a water/methanol mixture, and determination was carried out by LC-MS/MS. Flunixin was detected using positive electrospray ionization in selected reaction monitoring (SRM). Estimated limit of quantification of the method was 0.5, 0.5, 0.25, and 0.05 μg/kg for the liver, kidney, muscle, and fat, respectively. The method was validated in animal tissues in terms of selectivity, linearity, trueness, precision, decision limit (CCα), and detection capability (CCβ). All the trueness values fell within a range between 73.6 and 84.8 %. Precision values for all levels of concentration tested showed excellent relative standard deviation (RSD < 15 %). The CCα and CCβ values have been established for each tissue. A relatively rapid and sensitive LC-MS/MS method for the quantitative determination of flunixin in different animal tissues was developed and validated. The method is suitable for monitoring the flunixin residues in animal tissues.

Rapid detection of antimicrobial residues in poultry: A consequence of non-prudent use of antimicrobials

A cross sectional survey was conducted in three major poultry markets at Enugu Urban area of Enugu State, South Eastern, Nigeria, to determine the prevalence of antimicrobial residues in commercial broiler birds using Premi? Test Kit. Antimicrobials generally due to their availability over the counter and non-enforcement of legislature on irrational use are often abused in both veterinary and medical practice in most developing countries. The misuse and overuse of these drugs lead to deposition of violative levels of antimicrobial residues in animal tissues meant for human consumption with several health consequences when consumed. Premi? Test like all other microbiological tests is based on the principle of inhibition and was able to detect antibiotic residues in 42 (60%) of the 70 sampled commercial birds from three major poultry markets in the study area. It detected also residues in 90 out of the 280 different organ matrices made up of 70 samples of each organ, of which Kidney was the highest at 48.6%, Gizzard (30.1%), liver (25.8%), and muscle (24.3%) in that order. No association was found between the occurrence of antibiotic residues and the location of the poultry markets sampled at P

Development and Validation of Electromigration Technique for the Determination of Lincomycin and Clindamycin Residues in Poultry Tissues

An analytical method to quantify lincosamide residues (lincomycin and clindamycin) in poultry tissue samples by micellar electrokinetic capillary chromatography combined with UV detection has been developed and validated. The clean-up procedure with a simple deproteinization and solid-phase extraction was successfully applied for the determination of antibiotics in different edible tissue samples of poultry. The electrophoretic separation was achieved using an uncoated fused silica capillary (57 cm × 75 μm i.d.), voltage 16 kV, temperature 22 °C, and the optimal wavelength at 200 nm. The running buffer consisting of 10 mM sodium tetraborate decahydrate (pH 9.3) and 25 mM sodium dodecylsulfate was employed. The validation of the method was performed by evaluation of the analytical parameters: linearity, specificity, precision, and accuracy with limits of detection of 13.2 and 18.5 ng/g, for clindamycin and lincomycin, respectively. Good intermediate precision was obtained with relative standard deviation values less than 12 %. The mean recoveries were 91.2 and 87.7 % for lincomycin and clindamycin, respectively. The obtained results confirm that the proposed method is capable to identify and quantify of lincosamide residues in animal tissues far below maximum residue limit values and could be suitable for routine application in commercial of poultry samples.

Monoclonal Antibody-Based Fluorescence Polarization Immunoassay for High Throughput Screening of Furaltadone and its Metabolite AMOZ in Animal Feeds and Tissues

A simple and fast homogeneous fluorescent polarization immunoassay (FPIA) was developed for the determination of furaltadone and its metabolite 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ). Monoclonal antibody with high cross-reactivity to furaltadone and the nitrophenyl derivative of AMOZ (NPAMOZ) were produced against a novel immunogen and the effects of several synthesized tracers on FPIA sensitivity studied. The proposed FPIA, using an optimum antibody and tracer pair, had an IC₅₀ of 4.3 µg L⁻¹ and limit of detection at 0.6 µg L⁻¹ for furaltadone, and 2.7 µgL⁻¹ and 0.3 µg L⁻¹ for NPAMOZ. Recoveries of furaltadone from animal feeds by FPIA ranged from 79.6 to 87.7%, while recoveries of AMOZ from animal tissues ranged from 72.9 to 83.1%. Good correlation (R>0.99) between the results of this FPIA and a standard analytical method was obtained. The FPIA does not require separation or washing steps and the total time required for equilibrium of the antibody-tracer interaction is only 10 min. These results indicated that the proposed FPIA offers great potential and utility for the high throughput screening of furaltadone residues in animal feed and its metabolite AMOZ residues in animal tissues.

Simultaneous determination of cyromazine and dicyclanil in animal edible tissues using UPLC-MS/MS

Cyromazine and dicyclanil are both used as insect growth regulators. This paper describes an easy and innovative simultaneous extraction method for residues of cyromazine and dicyclanil in food of animal origin, and a confirmation procedure using UHPLC-MS/MS. The sample was extracted, deproteinised by 1% trichloracetic acid in water-acetonitrile, followed by selective defatting using hexane based on the degree of matrix complexity; cleaned-up on an mixed-mode cation exchange (MCX) cartridge; and quantified by using matrix-matched calibration. The mean recoveries were all between 62.0% and 99.2%, and the RSDs were all below 9.94%. The present method was rapid, sensitive and reliable, which was applied to the quantitative analysis of these residues in animal tissues.

Determination of Zeranol and its Metabolites in Bovine Muscle Tissue with Gas Chromatography-Mass Spectrometry

Abstract This paper describes the quantitative method of determination of chosen substances from resorcylic acid lactones group: zeranol, taleranol, α-zearalenol, β-zearalenol, and zearalanone in bovine muscle tissue. The presented method is based on double diethyl ether liquid-liquid extraction (LLE), solid phase extraction (SPE) clean up, and gas chromatography mass spectrometry (GC-MS) analysis. The residues were derivatised with a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide, ammonium iodide, and DL-dithiothreitol (1,000:2:5, v/w/w). The GC-MS apparatus was operated in positive electron ionisation mode. The method was validated according to the European Union performance criteria pointed in Decision Commission 2002/657/EC. The average recoveries of all analytes at 1 μg kg-1 level were located between 83.7% and 94.5% values with the coefficients of variation values &lt;25%. The decision limits (CCα) and detection capabilities (CCβ) for all analytes ranged from 0.58 to 0.82 μg kg-1 and from 0.64 to 0.94 μg kg-1, respectively. The procedure has been accredited and is used as a screening and confirmatory method in control of hormone residues in animal tissues.

Prediction of Gas Chromatography‐Mass Spectrometry Retention Times of Pesticide Residues by Chemometrics Methods

A quantitative structure‐retention relationships (QSRRs) method is employed to predict the retention time of 300 pesticide residues in animal tissues separated by gas chromatography‐mass spectroscopy (GC‐MS). Firstly, a six‐parameter QSRR model was developed by means of multiple linear regression. The six molecular descriptors that were considered to account for the effect of molecular structure on the retention time are number of nitrogen, Solvation connectivity index‐chi 1, Balaban Y index, Moran autocorrelation‐lag 2/weighted by atomic Sanderson electronegativity, total absolute charge, and radial distribution function‐6.0/unweighted. A 6‐7‐1 back propagation artificial neural network (ANN) was used to improve the accuracy of the constructed model. The standard error values of ANN model for training, test, and validation sets are 1.559, 1.517, and 1.249, respectively, which are less than those obtained reveals by multiple linear regressions model (2.402, 1.858, and 2.036, resp.). Results obtained the reliability and good predictability of nonlinear QSRR model to predict the retention time of pesticides.

Support vector regression based QSPR for the prediction of retention time of pesticide residues in gas chromatography–mass spectroscopy

In this study, a quantitative structure–property relationship (QSPR) method was employed to predict the retention time (tR) of 368 pesticide residues in animal tissues separated by gas chromatography–mass spectroscopy (GC–MS). The variable selection method of genetic algorithm-partial least squares (GA-PLS) was employed to select most favorable subset of descriptors. The six descriptors selected using GA-PLS were used as inputs of PLS, ANN and SVM to predict the retention times. These descriptors are: number of nitrogen atoms, solvation connectivity index‐Chi 1, Balaban Y index, Moran autocorrelation‐lag 2/weighted by atomic Sanderson electronegativity, total absolute charge and radial distribution function-6.0/unweighted. The correlation coefficients, R, between experimental and predicted tR for the prediction set by PLS, ANN and SVM are 0.907, 0.963 and 0.985 respectively. Results obtained reveal the reliability and good predictability of nonlinear QSPR model to predict the retention time of pesticides. Comparison between the values of statistical parameters reveals the superiority of the SVM model over PLS and ANN ones.

A determinative and confirmatory method for ceftiofur metabolite desfuroylceftiofur cysteine disulfide in bovine kidney by LC–MS/MS

Ceftiofur is a cephalosporin β-lactam antibiotic widely used for treating certain bacterial infections in beef and dairy cattle. The regulatory HPLC–UV method for ceftiofur residues in animal tissues is time consuming and non-specific. Additionally, because the regulatory method involves chemical reactions to convert the metabolites into a single moiety, it is virtually impossible to incorporate the procedure into a multi-residue method. Ceftiofur residue violations in beef and dairy cattle have been frequently reported and therefore an improved method is needed. Herein we report a rapid and sensitive LC–MS/MS method for the determination and confirmation of ceftiofur metabolite, desfuroylceftiofur cysteine disulfide (DCCD), in bovine kidney tissue. The new method utilizes a simple extraction with phosphate buffer followed by SPE cleanup. A deuterated internal standard was synthesized and used for quantitation. The matrix-based calibration curve was linear from 25 to 2000ng/g. The average accuracy for control kidney samples from six different sources fortified at 50–1000ng/g was 97.7–100.2% with CV≤10.1%. The limit of confirmation was 50ng/g.

Ion Exchange Solid Phase Extraction Coupled with Liquid Chromatography Tandem Mass Spectrometry for Determination of 8 Classes of Non-steroidal Anti-inflammatory Drugs Residues in Animal Tissues

An ultraperformance liquid chromatography tandem mass spectrometric(UPLC-MS/MS) method was established for the simultaneous determination of residues of 8 classes of 14 non-steroidal anti-inflammatory drugs(NSAIDs) in animal tissues.The samples were extracted with acetonitrile-ethyl acetate(1 ∶ 1,V/V),defatted with n-hexane,and then purified by MCX solid phase extraction(SPE) cartridge.Analysis was carried out on UPLC-ESI-MS/MS working with multiple reaction monitoring mode with polarity switching.Limits of detection were between 3.0 μg/kg and 10.0 μg/kg,and limits of quantification were between 10.0 μg/kg and 25.0 μg/kg.At spiked levels of 10.0-1000.0 μg/kg,in bovine and swine muscle the recoveries were 62.9%-108.4% and 63.4%-117.0%,respectively,and relative standard deviations(RSD) of the method were less than 10% and 9%,respectively.

Residue control of coccidiostats in food of animal origin in Poland during 2007–2010

Coccidiostats are widely used as feed additives to prevent coccidiosis. The off-label use of anticoccidials or feeding non-target animals with cross-contaminated feedingstuffs may result in the occurrence of coccidiostat residues in animal tissues and eggs. In EU countries, food of animal origin is subjected to official control of residues according to Council Directive 96/23/EC. In Poland, within the framework of the National Residue Control Plan, 3718 samples (3533 targeted and 185 suspect) of animal liver, eggs, drinking water and feed were tested for coccidiostats between 2007 and 2010. Violative residues of nicarbazin, lasalocid, maduramicin, salinomycin, semduramicin and robenidine were detected in 77 food samples (53 samples of chicken liver, 23 samples of eggs and 1 sample of turkey liver). A high percentage (31%) of non-compliant feed samples collected during follow-up investigations was observed, which confirms that feed cross-contamination may be the reason of the occurrence of coccidiostat residues in food.

Simultaneous determination of lincomycin and spectinomycin residues in animal tissues by gas chromatography-nitrogen phosphorus detection and gas chromatography-mass spectrometry with accelerated solvent extraction

A new multi-dimensional analytical method using gas chromatography-nitrogen phosphorus detection (GC-NPD) and gas chromatography-mass spectrometry (GC-MS) was developed for qualitative and quantitative measurement of lincomycin and spectinomycin residues in food animal tissues. This method is based on a new extraction procedure using accelerated solvent extraction (ASE). The analytes were extracted by phosphate buffer with trichloroacetic acid de-proteinisation and clean-up by C18 solid-phase extraction (SPE) adding dodecanesulfonic acid sodium salt as an ion-pair reagent. The eluted fraction was evaporated and derivatised with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) for GC-NPD analysis and GC-MS confirmation. Parameters for extraction pressure, temperature and cycle of ASE, clean-up, derivatisation and analysis procedure were optimised. The method was validated in muscle, kidney and liver of swine, bovine with a low concentration (limit of quantification) of 16.4 and 21.4 µg kg−1 for these two analytes using GC-NPD. For GC-MS, the limits of quantification were 4.1 and 5.6 µg kg−1, respectively. Spiked recoveries from levels of 20 to 200 µg kg−1 were found to be between 73% and 99% with a relative standard deviation (RSD) of less than 17% in GC-NPD. For GC-MS, levels from 5 to 20 µg kg−1 had between 70% and 93% with an RSD of less than 21%. This rapid and reliable method can be used for the characterisation and quantification of residues of lincomycin and spectinomycin in animal tissues.