Simple detection of residual enrofloxacin in meat products using microparticles and biochips

A simple and sensitive method for detecting enrofloxacin, a major veterinary fluoroquinolone, was developed. Monoclonal antibody specific for enrofloxacin was immobilised on a chip and fluorescent dye-labelled microparticles were covalently bound to the enrofloxacin molecules. Enrofloxacin in solution competes with the microparticle-immobilised enrofloxacin (enroMPs) to bind to the antibody on the chip. The presence of enrofloxacin was verified by detecting the fluorescence of enrofloxacin-bound microparticles. Under optimum conditions, a high dynamic range was achieved at enrofloxacin concentrations ranging from 1 to 1000 μg kg^?1. The limits of detection and quantification for standard solutions were 5 and 20 μg kg^?1 respectively, which are markedly lower than the maximum residue limit. Using simple extraction methods, recoveries from fortified beef, pork and chicken samples were 43.4–62.3%. This novel method also enabled approximate quantification of enrofloxacin concentration: the enroMP signal intensity decreased with increasing enrofloxacin concentration. Because of its sensitivity, specificity, simplicity and rapidity, the method described herein will facilitate the detection and approximate quantification of enrofloxacin residues in foods in a high-throughput manner.     

Development of an Immunochromatographic Test Strip for Rapid Simultaneous Detection of Enrofloxacin and Ofloxacin in Tissue of Chicken Muscle and Pork

ENR and OFL are the most consumed quinolones on livestock in China. In this work, we developed a rapid immunochromatographic lateral flow test strip for simultaneous detection of the residues of enrofloxacin and ofloxacin in chicken muscle and pork. We screened an anti-ENR and OFL monoclonal antibody. The IC₅₀ of anti-ENR and anti-OFL were 6.67 and 7.13 ng/ml, respectively. The present immunochromatographic lateral flow test strip is a one-step assay and required much less professional personnel and experimental instruments. In the present study, the decision limit (CCα) of the test strip was calculated to be 0.089 ng/mL and detection capability (CCβ) was 0.217 ng/mL with the scanner. The limit of detection was estimated to be 10 ng/mL. According to parallel HPLC analysis with 47 blind samples, coincidence rate is 100 % when the contents of ENR and OFL were more than 10 ng/mL. Results indicated that the strip test we developed had good reliability, and the strip test gave neither false positive nor false negative results. It will provide results within 20 min without special equipment. Therefore, the test strip is very useful as a screening method for semi-quantitative or qualitative detection of enrofloxacin and ofloxacin in chicken muscle and pork.     

Detection of herbicides in drinking water by surface-enhanced Raman spectroscopy coupled with gold nanostructures

This study aimed to use surface-enhanced Raman spectroscopy (SERS) to detect herbicide residues (atrazine and arsenic trioxide) in drinking water. Gold-coated nanosubstrates were used in the SERS measurements to acquire enhanced Raman signals of herbicides in drinking water. Compared with the control, characteristic patterns of SERS spectra were distinguishable for atrazine at 3 ppb and arsenic trioxide at 1 ppb. Partial least squares analysis was used to develop quantitative models for detection of two herbicides in drinking water and calibration curves were plotted with R² of 0.988 and 0.991 for atrazine and arsenic trioxide, respectively. The study of limit of detection (LOD) demonstrates that at 99.86 % confidence interval, SERS can detect both herbicides at 0.1 ppm in drinking water. Satisfactory recoveries were obtained for samples with concentration at and higher than the LOD (92.3–119.3 % for atrazine and 88.2–102.1 % for arsenic trioxide). These results demonstrate that SERS coupled with gold nanostructures holds great potential for rapid detection of herbicide residues and other chemical contaminants in drinking water.     

Investigation of enrofloxacin residues in broiler tissues using ELISA and LC-MS/MS

This study investigated the efficiency of an enrofloxacin ELISA test kit to detect the presence of enrofloxacin residues in broiler tissues compared with LC-MS/MS. Broiler tissues from 72 samples consisting of 60 breast muscle, six pools of livers (500 g each) and six pools of kidneys (500 g each) were obtained from six different slaughterhouses. Breast muscle from 10 carcasses and pools of livers and kidneys from approximately 200 carcasses of the same flock were collected from each slaughterhouse. ELISA and HPLC were used to identify and quantify the contamination of the samples with enrofloxacin. A total of 72% of the analysed samples contained enrofloxacin residues detected by the ELISA and 22.2% were detected by LC-MS/MS. The mean values of enrofloxacin contamination found in chicken breast by ELISA and HPLC were 8.63 and 12.25 μg kg^–1, respectively. None of the samples exceeded the maximum limit of 100 μg kg^–1 by both methods set by the European Union as well as the Brazilian Agriculture Ministry. All positive samples for enrofloxacin residues detected by LC-MS/MS were also positive by ELISA. These data confirm the efficiency of the ELISA test, and suggest its use as a screening method for enrofloxacin residues in poultry tissues due to its quick results, low price and ease of applicability.     

A sensitive chromatographic strip test for the rapid detection of enrofloxacin in chicken muscle

A sensitive colloidal gold immunochromatography assay using a specific monoclonal antibody was developed for the rapid detection of enrofloxacin (ENR) residues in chicken muscles. Anti‐ENR antibodies with high sensitivity and specificity are generated by immunising BALB/c mice with well‐characterised ENR‐bovine serum albumin conjugate. An orthogonal L₉(3)³ test was designed, and various parameters that influenced the assay performance were investigated and optimised. Under the optimised conditions, the cut‐off limits of semi‐quantitative test strips for ENR were found to be 3?ng/mL in phosphate‐buffered saline and 8?µg/kg in chicken muscle. The ENR test strips showed a 6% cross‐reactivity with ciprofloxacin, 3% with norfloxacin, less than 1% with ofloxacin and sarafloxacin and 0.1% with the other eight fluoroquinolones including enoxacin, difloxacin, danofloxacin, pefloxacin, lomefloxacin, sparfloxacin, oxolinic acid and flumequine. Consistent results are produced from the parallel analysis of ENR‐contaminated chicken muscle extracts using test strips and ELISA.     

Rapid determination of gizzerosine in fish meals using microchip capillary electrophoresis with laser-induced fluorescence detection

ABSTRACT

Sensitive detection of gizzerosine, a causative agent for deadly gizzard erosion in chicken feeds, is very important to the poultry industry. In this work, a new method was developed based on microchip capillary electrophoresis (MCE) with laser-induced fluorescence (LIF) detection for rapid analysis of gizzerosine, a biogenic amine in fish meals. The MCE separation was performed on a glass microchip using sodium dodecyl sulfate (SDS) as dynamic coating modifier. Separation conditions, including running buffer pH and concentration, SDS concentration, and the separation voltage were investigated to achieve fast and sensitive quantification of gizzerosine. The assay proposed was very quick and could be completed within 65 s. A linear calibration curve was obtained in the range from 0.04 to 1.8 μg ml^–1 gizzerosine. The detection limit was 0.025 μg ml^–1 (0.025 mg kg^–1), which was far more sensitive than those previously reported. Gizzerosine was well separated from other endogenous components in fish meal samples. Recovery of gizzerosine from this sample matrix (n = 3) was determined to be 97.2–102.8%. The results from analysing fish meal samples indicated that the present MCE-LIF method might hold the potential for rapid detection of gizzerosine in poultry feeds.     

Detection of Pesticides in Fruits by Surface-Enhanced Raman Spectroscopy Coupled with Gold Nanostructures

There is growing concern in recent years for consumers about contamination of pesticides in fruits due to increasing use of pesticides in fruits. The objective of this study was to use surface-enhanced Raman spectroscopy (SERS) to detect and characterize pesticides extracted from fruit surfaces. Gold-coated SERS-active nanosubstrates were used for SERS measurement. Three types of pesticides (carbaryl, phosmet, and azinphos-methyl) widely used in apples and tomatoes were selected. Significantly enhanced Raman signals of pesticides were acquired by SERS from the extract of fruit samples and exhibited characteristic patterns of the analytes. Multivariate statistical methods such as partial least squares and principal component analysis were used to develop quantitative and qualitative models. SERS was able to detect all three types of pesticides extracted from fruit samples at the parts per million level. The study of detection limit demonstrated that at 99.86% confidence interval, SERS can detect carbaryl at 4.51 ppm, phosmet at 6.51 ppm, and azinphos-methyl at 6.66 ppm spiked on apples; and carbaryl at 5.35 ppm, phosmet at 2.91 ppm, and azinphos-methyl at 2.94 ppm on tomatoes. Most of these detection limits meet the maximum residue limits established by Food and Agriculture Organization of the United Nations and World Health Organization. Satisfactory recoveries (78–124%) were achieved for samples with concentrations at and larger than the detection limit. These results demonstrate that SERS coupled with novel SERS-active nanosubstrates is a rapid, sensitive, and reliable method for detection and characterization of chemical contaminants in foods.     

Reproducible Molecularly Imprinted QCM Sensor for Accurate,Stable, and Sensitive Detection of Enrofloxacin Residue in Animal-Derived Foods

This study describes the development of a novel reproducible molecularly imprinted quartz crystal microbalance (QCM) sensor for the accurate and sensitive analysis of the residue of enrofloxacin (ENRO) in animal-derived foods. This proposed sensor was easily fabricated by directly immobilizing molecularly imprinted polymer (MIP) of ENRO on the surface of a QCM Au chip, which combined the advantages of selective recognition from the MIP with the high sensitivity and portability of a QCM sensor. The parameters in the fabrication and measurement process were optimized and discussed in detail. It was verified that the MIP-modified QCM Au chip performed favorably for the detection of ENRO residue in common animal-derived food products and demonstrated acceptable accuracy (recovery: pure milk 77.2–84.2%, egg 77.3–85.6%, chicken muscle 73.5–89.1%, pork 74.7–85.8%), precision (relative standard deviation (RSD, n = 3), pure milk 2.9–8.0%, egg 2.9–6.4%, chicken muscle 3.4–6.8%, pork 2.2–4.7%), and sensitivity (limit of detection, pure milk 0.31 μg L^?1, egg 0.44 μg kg^?1, chicken muscle 0.54 μg kg^?1, pork 0.57 μg kg^?1). The MIP-modified QCM Au chip for sensing ENRO was portable, could be stored for an extended period of time, and reused for more than 30 analysis cycles with a response attenuation of 7.8%. These results have demonstrated that the proposed MIP QCM sensor presents an accurate, sensitive, rapid, and low-cost methodology for ENRO residue detection in animal foods. This research is very promising for the development of novel effective devices applied to the detection of various contaminants in the field of food safety.     

Development and Optimization of a SERS Method for On-site Determination of Nitrite in Foods and Water

A rapid, sensitive, and selective detection method of nitrite in foods and water was established by surface-enhanced Raman spectroscopy (SERS) based on the diazo reaction of nitrite with p-nitroaniline and 1-naphthylamine in acidic solution. The azo dye (4-(4-nitrophenyldiazenyl)naphthalene-1-aminium, NNA), which was derived from the diazo reaction, was determined by SERS using citrate-coated silver nanoparticles (AgNPs) as SERS substrates. The concentrations of nitrite in samples were finally calculated from the intensities of SERS signals generated by NNA in the testing solutions. By using the present method combined with a portable miniature Raman spectrometer, on-site determination of nitrite could be performed easily and efficiently. The effects of several experimental parameters on the intensity of SERS signals, such as the volume of AgNP solution and the mixing time of AgNPs and NNA, were investigated. The linear range of the method was 0.1–10.0 mg L^?1. The limits of detection (LODs) were 0.01, 0.03, and 0.05 mg L^?1 at 720, 1,459, and 1,609 cm^?1, respectively. The method was applied to the determination of nitrite in real food and water samples, with recoveries in the range of 86.9–103.4 % and relative standard deviations (RSDs) less than 9.64 %.     

Detection of Omethoate Residues in Peach with Surface-Enhanced Raman Spectroscopy

In this work, surface-enhanced Raman spectroscopy (SERS) was used with silver colloid substrate for rapid detection of omethoate (an organophosphate pesticide) in standard solution and peach extract. The findings demonstrated that the characteristic wavenumber of the pesticide could be precisely identified using the SERS method. The calibration curve was plotted between concentrations and Raman intensities of the target peak at 1649 cm^?1 for the peach extract and at 1647 cm^?1 for the standard solution. The coefficients of determination (R²) of 0.9829 and 0.98 were obtained for standard solution and for peach extract, respectively. The calculated limits of detection for omethoate in standard solution and in peach extracts were 0.001 mg L^?1 and 0.01 mg kg^?1, respectively. This study revealed that the proposed method could be used for the analysis of trace contaminants like omethoate in multifaceted food matrices.     

Potential of SERS for rapid detection of melamine and cyanuric acid extracted from milk

Melamine, a nitrogen-rich chemical, was implicated in the pet and human food recalls in 2007 and in the global food safety scares in 2008 involving milk and other milk-derived products. In this study, we investigated the feasibility of using surface-enhanced Raman spectroscopy (SERS) coupled with SERS-active gold substrates for rapid detection of trace amounts of melamine and its analogue (that is, cyanuric acid) in liquid milk. Raman signals of tested samples were significantly enhanced by SERS. The identification limit for SERS using gold substrate can reach 2 ppm of melamine in liquid milk. Partial least squares (PLS) models were established for the quantification of melamine in liquid milk by SERS: R = 0.90, RMSEP = 1.48 × 10⁻⁵. Our results demonstrate that rapid detection of melamine in milk can be achieved by SERS; while detection of cyanuric acid in milk remains a challenging task due to rapid enol-keto tautomerism of cyanuric acid. The SERS method is faster and simpler than other traditional methods, and requires minimum sample preparation. These results demonstrate that SERS could be used to detect food contaminants such as melamine in foods and food ingredients quickly and accurately.     

A HPTLC-Fluorescent Densitometry Assay for Simultaneous Detection of Enrofloxacin and Ciprofloxacin in Broiler Chicken Tissues

A high-performance thin-layer chromatographic (HPTLC) method was developed and validated according to the protocol on Validation of Analytical Procedures: methodology, Veterinary International Cooperation on Harmonization for simultaneous determination of enrofloxacin and ciprofloxacin in broiler chicken tissues viz. liver, kidney, muscle-breast, muscle-thigh, and skin. Chromatography was performed on thin-layer chromatography (TLC) silica gel 60F₂₅₄, aluminum sheets (10 × 10 cm) by Camag Linomat-5 applicator. The isolation of enrofloxacin and ciprofloxacin was attained by two development stages, initially with diethyl ether followed by dichloromethane/methanol/aqueous ammonia/acetonitrile (2:3:2:3) as mobile phase. The developed TLC plates were exposed to hydrochloric acid fumes and fluorescent densitometric evaluation was performed at 366 nm using Camag TLC Scanner-3 with WinCAT 1.4.4 software. The R_F value was 0.60 and 0.44 for enrofloxacin and ciprofloxacin, respectively. The detection limits were 2 and 3 ng/band for enrofloxacin and ciprofloxacin, respectively, and 5 ng/band as limit of quantification for both the compounds. Enrofloxacin and ciprofloxacin showed wide linear range from 2 to 110 ng/band and 3 to 110 ng/band with high correlation coefficient values of 0.99997 and 0.99990 for peak area, respectively. An exemplary precision was observed for individual compounds. The percent recovery for ciprofloxacin was 82.0–86.8% when compared to enrofloxacin 83.4–90.3%. The observed results were within the acceptable values as a function of the analyte concentration, suggested by the Association of Official Analytical Chemists peer-verified methods program. The present validated HPTLC method can be used as screening technique for antibiotics residue monitoring program in broiler chicken tissues to promulgate food safety.     

LC-MS/MS-based determination of chloramphenicol,thiamphenicol, florfenicol and florfenicol amine in poultry meat from the Punjab-Pakistan

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry-based confirmatory method was redeveloped and validated for the simultaneous determination of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscles. The analytes were extracted from minced chicken muscle with acetonitrile and ammoniated water mixture. A second extraction with ethyl acetate was followed by evaporation and dissolution of the residue in ammoniated methanol before defatting with n-hexane. Finally, the extract was further cleaned up by dispersive solid phase extraction using C-18 end-capped dispersive material. The validation protocol was adapted from the European Commission Decision 2002/657/EC and all the performance characteristics were successfully satisfied. The recoveries of all the analytes were found to be in the range of 86.4–108.1% and the precision values, within day and between days, ranged from 2.7% to 11% and 4.4% to 16.3%, respectively. The method was tested in various incurred samples and applied to analyse a wide range of random poultry market samples (n = 120) collected from three cities of the Punjab, Pakistan. Chloramphenicol and florfenicol residues were detected at low levels in less than 11% of the samples. Chloramphenicol was detected only in 4 samples with the concentration range of 0.17–0.477 µg kg^–1, whereas the levels of florfenicol/florfenicol amine residues detected in 9 samples ranged from 8.7 to 32.8 µg kg^–1. Moreover, most of the florfenicol residues were identified as tissue bound, extractable only after strong acid hydrolysis.     

Rapid Analysis of Bitertanol in Agro-products Using Molecularly Imprinted Polymers-Surface-Enhanced Raman Spectroscopy

In this work, we first reported molecularly imprinted polymers-surface-enhanced Raman spectroscopy (MIP-SERS) for rapid detection and quantification of bitertanol in cucumber and peaches. In order to remove interference-mitigation effects and avoid template leakage, MIPs were successfully synthesized based on triadimefon molecules as the dummy template, methacrylic acid (MAA) as a functional monomer, trimethylolpropane trimethacrylate (TRIM) as a cross-linker, and 2,2-azobis-isobutyronitrile (AIBN) as an initiator. Static adsorption experiments and Scatchard analysis were then conducted and results showed that the synthesized MIPs could rapidly and selectively adsorb and separate bitertanol from cucumber and peaches due to their predetermined recognition sites. The capacity of MIPs for absorbing bitertanol (～ 2.21 mg/g) was approximately 1.5 times that of non-imprinted polymers (NIPs) (～ 1.55 mg/g). Gold nanoparticles (AuNPs) synthesized by sodium citrate reduction method were validated as a suitable SERS colloid for enhancing Raman signals. SERS spectral peaks (760, 985, 1190, and 1279 cm^?1) were used to develop quantitative analysis based on partial least-squares regression (PLSR) for bitertanol in cucumber and peaches. The LODs for this method were 0.041 and 0.029 mg/kg in cucumber and peaches, respectively. The entire analysis process required 15 min or less. More importantly, the MIP-SERS system provided not only a rapid, sensitive, and reliable method for bitertanol detection, but also a routine for overcoming the interference-mitigation effects in the SERS technology.     

Determination of 6-Benzylaminopurine and Hg<Superscript>2+</Superscript> in Bean Sprouts and Drinking Mineral Water by Surface-Enhanced Raman Spectroscopy

A novel method for the determination of 6-benzyladenine (6-BA) in bean sprouts and Hg²⁺ in drinking mineral water by surface-enhanced Raman spectroscopy (SERS) was described. 6-BA exhibits obvious SERS signal by using the substrate of silver nanoparticles (AgNPs), and the presence of Hg²⁺ could cause the decrease of SERS signal of 6-BA. The effects of type of aggregation agent, type and level of pH buffer solution, amount of AgNPs, mixing time, concentration of 6-BA, and reaction time on the SERS signals were investigated. Under the optimized experimental conditions, good linear responses were obtained for 6-BA and Hg²⁺ in the concentration ranges of 10–200 μg L^?1 and 5–200 μg L^?1, respectively. By the present method, the limits of detection (LODs) for the determination of 6-BA and Hg²⁺ are 3.3 and 0.20 μg L^?1, and the recoveries of 6-BA and Hg²⁺ in spiked samples are 85.5–113.0 % and 98.2–111.5 %, respectively.     

Determination of Benzalkonium Homologues and Didecyldimethylammonium in Powdered and Liquid Milk for Infants by Hydrophilic Interaction Liquid Chromatography–Mass Spectrometry

In this study, a hydrophilic interaction liquid chromatography–mass spectrometry (HILIC-MS/MS) method for the determination of benzalkonium (BAC) homologues and didecyldimethylammonium (DDAC) was developed. A satisfactory chromatographic separation of BAC homologues and DDAC was achieved using, as mobile phase, acetonitrile–aqueous 50 mM ammonium formate (pH 3.2) (93?+?7 v/v) at 0.3 mL min^?1. The elution order of BAC homologues was from benzyldimethylhexadecylammonium chloride (C₁₆-BAC) to benzyldimethyldecylammonium chloride (C₁₀-BAC), the exact opposite with respect to separation using reversed liquid chromatography. The instrumental method was successfully applied to powdered and liquid milk for infants (about 50 samples). From powdered milk samples, BAC and DDAC were extracted using 5 % formic acid in methanol for 60 min at 60 °C in an ultrasonic bath; after dilution with water and 5 % NH₄OH solution, a purification step using a weak cationic exchange column was performed. Satisfactory limit of detections (LODs) were achieved, below 1.0 μg kg^?1 and 0.05 μg L^?1 for powdered and liquid milk for infants, respectively. No sample was free of BAC homologues and DDAC, and in one powdered milk sample, the contamination level exceeded 500 μg kg^?1, the maximum level recommended by the Standing Committee on the Food Chain and Animal Health for food and feed.     

A Highly Sensitive Aptasensor for Sulfamethazine Detection Using an Enzyme-Linked Aptamer Assay

To detect sulfamethazine (SMZ) residues in edible animal products, a colorimetric aptasensor that is based on indirect competitive enzyme-linked aptamer assay (ELAA) is developed in this study. This ELAA uses SMZ-specific aptamers as recognition elements and enzyme as the signal readout element. The detection sensitivity and the specificity of the ELAA were investigated. Our results showed that the indirect competitive ELAA had a ultrasensitivity with a low detection limit of 0.05 ng mL^?1. In addition, the proposed indirect competitive ELAA exhibited an 50% inhibition concentration value of 0.64 ng mL^?1 for SMZ, and recoveries up to 80.5–92.3% with coefficients of variation of less than 10%. Furthermore, when the ELAA was used for incurred samples by detecting SMZ in chicken muscles, the results obtained by the ELAA methods showed a good agreement with those obtained by established TRFIA and HPLC methods. These results demonstrate that the proposed indirect competitive ELAA can be efficiently used with good accuracy and precision, providing a simple method for the detection of SMZ residue in animal tissues.     

Detection and quantification of 47 antibiotic residues in farmed European sea bass (Dicentrarchus labrax) using a multi-class and multi-residue UHPLC-MS/MS method

In the present study, a multi-class multi-residue method for the simultaneous detection and determination of antibiotics in European sea bass (Dicentrarchus labrax) was developed and validated. The method based on UHPLC-MS/MS proved to be a rapid, highly selective and sensitive tool, requiring minimum sample preparation, for screening and detection of 47 compounds from eight different classes. The validation was performed according to EU regulation 2002/657/EC, proving the method’s suitability for application in routine analysis. The method was applied to the analysis of 30 samples of farmed European sea bass purchased in different supermarkets in Portugal. Antibacterial residues were detected in 6 of the 30 analysed samples, namely enrofloxacin and oxytetracycline, in concentrations ranging from 0.1 to 12 µg kg^?1.     

Detection and differentiation of Salmonella serotypes using surface enhanced Raman scattering (SERS) technique

This research was conducted to prove that developed silver biopolymer nanoparticle substrate for surface enhanced Raman scattering (SERS) technique could detect and differentiate three different serotypes of Salmonella. Nanoparticle was prepared by adding 100 mg of silver nitrate to a 2 % polyvinyl alcohol solution, then adding 1 % trisodium citrate to reduce silver nitrate and produce silver encapsulated biopolymer nanoparticles. Then, nanoparticle was deposited on a stainless steel plate and used as SERS substrate. Fresh cultures of Salmonella typhimurium, Salmonella enteritidis and Salmonella infantis were washed and suspended in 10 mL of sterile deionized water. Approximately 5 μl of the bacterial suspensions were placed on the substrate individually and exposed to 785 nm laser excitation. SERS spectral data were recorded between 400 and 1,800 cm^?1. SERS signals were collected from 15 different spots on the substrate for each sample. PCA model was developed to classify Salmonella serotypes. PC1 identified 92 % of the variation between the Salmonella serotypes, and PC2 identified 6 % and in total 98 % between the serotypes. Soft independent modeling of class analogies of validation set gave an average correct classification of 92 %. Comparison of the SERS spectra of Salmonella serotypes indicated that both isolates have similar cell walls and cell membrane structures which were identified by spectral regions between 520 and 1,050 cm^?1. However, major differences were detected in cellular genetic material and proteins between 1,200 and 1,700 cm^?1. SERS with silver biopolymer nanoparticle substrate could be a promising tool in pathogen detection and it would potentially be used to classify them.     

Simultaneous Determination of Cyadox and Its Metabolites in Chicken Tissues by LC-MS/MS

A specific and sensitive LC-MS/MS method was firstly established for the simultaneous extraction and determination of cyadox and its three main metabolites—1,4-bisdesoxycyadox, 4-desoxycyadox, and quinoxaline-2-carboxylic acid—in chicken muscle, liver, kidney, and fat tissues. Samples were subjected to extraction using ethyl acetate and followed by acetonitrile–chloroform (1:4, v/v) and further purified by Oasis mixed mode anion exchange SPE cartridge. Analysis was performed on a C₁₈ column by detection with MS in multiple-reaction monitoring mode. A gradient elution program with 0.1 % formic acid solution, acetonitrile, and 1 % formic acid (adjusted to pH 8 with ammonia) was performed at a flow rate of 0.2 mL min^?1. The correlation coefficients (r) for each calibration curve are higher than 0.99 within the experimental concentration range. The recoveries of the four target analytes at three spiking levels of 2.5, 25 and 250 μg kg^?1 were between 74.5 and 93.8 %, with relative standard deviations less than 12 %. The decision limits (CCαs) of the four analytes in chicken edible tissues ranged from 0.3 to 1.5 μg kg^?1, and the detection capabilities (CCβs) were below 2.3 μg kg^?1. The developed method demonstrated a satisfactory applicability in incurred chicken tissue samples.