Determination of marker residues of quinoxaline-1,4-di-N-oxides and its prototype identification by liquid chromatography tandem mass spectrometry.

Quinoxaline-1,4-di-N-oxides (QdNOs), such as carbadox, olaquindox, mequindox, quinocetone, etc. are a class of antibacterial drugs. Prototype drugs residues can not be detected due to their rapid metabolism in animals. Quinoxaline-2-carboxylic acid (QCA) and 3-methyl-QCA (MQCA) are their common marker residues, so it has been always a challenge to trace the specific QdNOs drug used in food animal production. Herein, a liquid chromatography tandem mass spectrometry method was developed to determine QCA and MQCA, and meanwhile, the prototype drugs were identified by analyzing bis-desoxy QdNOs metabolites in single ion-pair monitoring mode. The method indicated that the average recoveries for QCA and MQCA were from 90 % to 105 % with relative standard deviations below 10 %, and the limits of quantification were 1.0 µg/kg. The limits of detection of five bis-desoxy QdNOs (qualitative markers) reached 0.5 µg/kg. This new analytical strategy can effectively solve the identification problem of QdNOs drugs in animal-derived food.

Hydrophilic-interaction-based magnetically assisted matrix solid-phase dispersion extraction of carbadox and olaquindox in feeds.

BACKGROUND: Carbadox and olaquindox have been banned from feeds since 1998 by the EU because of their mutagenic, photoallergic, and carcinogenic effects. Unfortunately, owing to their outstanding effect, they are frequently abused or misused in animal husbandry. There is an urgent need to develop a sensitive and reliable method for monitoring these drugs in animal feeds. RESULTS: This work reported a new method of hydrophilic-interaction-based magnetically assisted matrix solid-phase dispersion (MMSPD) extraction coupled with reversed-phase liquid chromatography-mass spectrometry for simultaneous determination of carbadox and olaquindox in animal feeds. 3-Trimethoxysilylpropyl methacrylate (γ-MAPS)-modified attapulgite (ATP) was crosslinked with γ-MAPS-modified iron(II,III) oxide (Fe3 O4 ), 1-vinyl-3-(butyl-4-sulfonate) imidazolium (VBSIm), acrylamide (AM), and N,N'-methylene-bis(acrylamide) (MBA) to synthesize ATP@Fe3 O4 @poly(VBSIm-AM-MBA) particles. The resultant particles were characterized by scanning electron microscopy, energy dispersive spectrometer, transmission electron microscopy, vibrating sample magnetometer, and Fourier transform infrared spectroscopy. Crosslinking of ATP into the magnetic particles has significantly increased the adsorption capacity of the particles. Under optimum conditions, the limits of detection (S/N = 3) were 0.3 µg kg-1 and 0.9 µg kg-1 for carbadox and olaquindox respectively. The intra-day and inter-day recoveries of the spiked targets in feed samples were in the range 83.5-98.3% with relative standard deviations of 1.0-8.3%. CONCLUSION: With a simplified procedure and a low amount of sample, the proposed hydrophilic-interaction-based MMSPD method is not only useful for the determination of carbadox and olaquindox in feeds but also holds great promise for the analysis of other polar targets in solid or semisolid matrices. © 2021 Society of Chemical Industry.

Novel Electrochemical Sensor Fabricated for Individual and Simultaneous Ultrasensitive Determination of Olaquindox and Carbadox Based on MWCNT-OH/CMK-8 Hybrid Nanocomposite Film.

A hybrid nanocomposite consisting of hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) and cube mesoporous carbon (CMK-8) was applied in this study to construct an MWCNT-OH/CMK-8/gold electrode (GE) electrochemical sensor and simultaneously perform the electro-reduction of olaquindox (OLA) and carbadox (CBX). The respective peak currents of CBX and OLA on the modified electrode increased by 720- and 595-fold relative to the peak current of GE. The performances of the modified electrode were investigated with electrochemical impedance spectroscopy, cyclic voltammetry, and differential pulse voltammetry. Then, the modified electrodes were used for the individual and simultaneous determination of OLA and CBX. The fabricated sensor demonstrated a linear response at 0.2-500 nmol/L in optimum experimental conditions, and the detection limits were 104.1 and 62.9 pmol/L for the simultaneous determination of OLA and CBX, respectively. As for individual determination, wide linear relationships were obtained for the detected OLA with levels of 0.05-500 nmol/L with LOD of 20.7 pmol/L and the detected CBX with levels of 0.10-500 nmol/L with LOD of 50.2 pmol/L. The fabricated sensor was successfully used in the independent and simultaneous determination of OLA and CBX in spiked pork samples.

Determination of olaquindox, carbadox and cyadox in animal feeds by ultra-performance liquid chromatography tandem mass spectrometry.

Olaquindox, carbadox, and cyadox are chemically synthesised antibacterial and growth-promoting agents for animals. At high doses they may exert mutagenicity and hepatic and adrenal toxicities in animals. Regrettably, these substances are frequently abused or misused when added into animal feeds. Thus, developing a sensitive and reliable method for simultaneous determination of olaquindox, carbadox, and cyadox in different kinds of animal feeds is crucially important for food safety monitoring. In this paper we optimised instrumental conditions, extraction solvents, solid phase extraction cartridges, and pH of the loading solvents on the Oasis HLB cartridge. Under the optimal conditions, mean recoveries ranged from 74.1 to 111%, and intra-day and inter-day variations were lower than 14.6% and 10.8%, respectively. The limits of quantification for olaquindox, carbadox, and cyadox were 0.05 mg kg-1, 0.10 mg kg-1, and 0.025 mg kg-1, respectively. The proposed method uses ultra-performance liquid chromatography tandem mass spectrometry and is sensitive and reliable for the simultaneous determination of olaquindox, carbadox, and cyadox in three kinds of animal feeds (specifically, mixed feed, concentrated feed, and additive premixed feed). This method has good precision, high sensitivity, and good reproducibility, and thus it can be used for convenient and accurate determination of olaquindox, carbadox, and cyadox in different kinds of animal feeds.

HPLC-MS/MS method validation for the detection of carbadox and olaquindox in poultry and swine feedingstuffs.

Carbadox (CBX) and olaquindox (OLA) were used in poultry and swine feed for growth promotion, to improve feed efficiency and increase the rate of weight gain. However, the use of these agents in feedingstuffs was prohibited because of concerns about their toxicity. Regulatory laboratories are required to have suitably validated analytical methods to ensure compliance with the ban. A quantitative and confirmatory method for determining the presence of CBX and OLA in poultry and swine feed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed, optimized, and validated. The analytes extraction was performed with a mixture of water and acetonitrile (1:1v/v) and cleanup with hexane and C18 (dispersive phase). The method was evaluated by the following parameters: specificity, linearity, matrix effect, decision limits (CCα), detection capability (CCß), accuracy, precision, limits of detection (LoD), limits of quantification (LoQ) and measurement uncertainty. The validated method presented a broad linear study range and no significant matrix effect. The limit of detection (LoD) was defined at 9 µg kg(-1) for CBX and 80 µg kg(-1) for OLA, and the limit of quantification (LoQ) was defined at 12 µg kg(-1) and 110 µg kg(-1) for CBX and OLA, respectively. The accuracy of the method was adequate for CBX and OLA. The recovery values found in the repeatability conditions were 99.41% for CBX and 104.62% for OLA. Under intralaboratory reproducibility conditions, the values were 98.63% for CBX and 95.07% for OLA. It was concluded that the performance parameters demonstrated total method adequacy for the detection and quantification of CBX and OLA in poultry and swine feedingstuffs.

Pharmaceuticals and other anthropogenic tracers in surface water: a randomized survey of 50 Minnesota lakes.

Water from 50 randomly selected lakes across Minnesota, USA, was analyzed for pharmaceuticals, personal care products, hormones, and other commercial or industrial chemicals in conjunction with the US Environmental Protection Agency's 2012 National Lakes Assessment. Thirty-eight of the 125 chemicals analyzed were detected at least once, all at parts per trillion concentrations. The most widely detected was N,N-diethyl-m-toluamide, present in 48% of the lakes sampled. Amitriptyline, a widely used antidepressant, was found in 28% of the lakes. The endocrine active chemicals bisphenol A, androstenedione, and nonylphenol were found in 42%, 30%, and 10% of the lakes, respectively. Cocaine was found in 32% of the lakes, and its degradation product, benzoylecgonine, was detected at 28% of the locations. Carbadox, an antibiotic used solely in the production of swine, was also present in 28% of the lakes sampled. The means by which these and other chemicals were transported to several of the remote lakes is unclear but may involve atmospheric transport.

Determination of carbadox and olaquindox metabolites in swine muscle by liquid chromatography/mass spectrometry.

This paper presents LC-MS/MS method that was developed for the simultaneous determination and confirmation metabolites of carbadox (desoxycarbadox, quinoxaline-2-carboxylic) and olaquindox (3-methylquinoxaline-2-carboxylic acid) residues in pig muscle tissues at concentrations ≤3.0µgkg(-1). Pig muscle tissues were deproteinated with meta-phosphoric acid in methanol and then were extracted with ethyl acetate:dichloromethane (50:50, v/v). The whole extracts were evaporated to dryness in rotary evaporator at 45°C, and dry residues were re-dissolved in 0.5% isopropanol in 1% acetic acid. The LC separation was performed on a C8 column with a gradient system consisting of isopropanol/water/acetic acid and methanol as the mobile phase. Additionally SelexION™ technology to reduce matrix effect was used. The decision limit (CCα) ranged from 1.04µgkg(-1) to 2.11µgkg(-1) and the detection capability (CCß) ranged from 1.46µgkg(-1) to 2.89µgkg(-1). The total recoveries were from 99.8% to 101.2%. The results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC.

Determination of Carbadox and metabolites of Carbadox and Olaquindox in muscle tissue using high performance liquid chromatography-tandem mass spectrometry.

A sensitive and robust LC-APCI-MS/MS method has been developed for the unambiguous detection and quantitative determination of the antimicrobial agent Carbadox, its metabolite quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid the major metabolite of Olaquindox. The method was aimed for application in the assaying of muscle tissue so the developed sample preparation scheme subjected samples to enzymatic digestion prior to the application of solid phase extraction clean-up. Subsequently the purified extracts were analyzed by reversed-phase LC-MS/MS in positive APCI and multiple reaction monitoring mode. The method was validated at a level of 1 µg/kg. The decision limits CCα and detection capability CCß ranged from 0.09 µg/kg to 0.24 µg/kg and from 0.12 µg/kg to 0.41 µg/kg, respectively. The accuracy and precision of the method were satisfactory. The recoveries ranged from 92% to 101% for the metabolites and from 60% to 62% for Carbadox, with coefficient of variances (CVs) less than 12%. The developed method proved efficient and straightforward allowing positive identification and quantitation of the target banned analytes and is thus suitable for application in residue control programmes and metabolism studies.

Progress on the development and single-laboratory validation of a high-performance liquid chromatographic method for the determination of carbadox and pyrantel tartrate in type B and C medicated feeds.

Carbadox, an antimicrobial agent, and pyrantel tartrate, an anthelmintic, are feed additives that are often used in combination in the United States. The current AOAC methods for these analytes are spectrophotometric, using standard addition techniques. These methods are labor-intensive and prone to variability as well as matrix interferences. Published methods for both analytes that use high-performance liquid chromatography were evaluated and a test method was developed. The method uses a water prewetting step to enhance extraction of pyrantel followed by extraction with acetonitrile-ethanol (50 + 50). Sample extracts are filtered through a glass fiber filter and purified using alumina solid-phase extraction columns. Chromatography is performed on a C18 column with a gradient mobile phase of dibutylamine acetate and acetonitrile. The data show that both analytes exhibit acceptable peak shape when a C18 column that is both acid- and base-deactivated is used. Linearity has been established and initial recovery studies on medicated swine feeds are promising.

A determinative and confirmatory method for residues of the metabolites of carbadox and olaquindox in porcine tissues.

Carbadox (CBX) and olaquindox (OLQ) are used in swine feed for growth promotion, to improve feed efficiency, increase the rate of weight gain, control swine dysentery and bacterial enteritis in young swine. In 1991, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) recommended maximum residue limits (MRLs) of 30 and 5mugkg(-1) in liver and muscle tissues of pigs, respectively, based on the concentration of, and expressed as, quinoxaline-2-carboxylic acid (QCA) as marker residue. In 1998, the European Commission (EC) banned the use of CBX and OLQ in food animal production together with four other feed additives, following reports that CBX and desoxycarbadox (DCBX) are suspect carcinogens and mutagens. In 2001, the sale of CBX was halted in Canada. In 2003, JECFA recommended the withdrawal of the previously recommended acceptable daily intake (ADI) and MRLs and concluded that QCA was not a suitable marker residue for CBX, based on new sponsor studies reporting that DCBX, the suspect carcinogen, persisted in animal tissues much longer than had previously been thought. This paper presents a very sensitive LC-MS/MS method that was developed by CFIA scientists for the simultaneous determination and confirmation of DCBX residues at concentrations >/=0.050 ngkg(-1) and QCA and mQCA residues at concentrations >/=0.50 ngkg(-1)in bovine muscle, pork liver and muscle tissues.

Matrix solid-phase dispersion extraction of carbadox and olaquindox in feed followed by hydrophilic interaction ultra-high-pressure liquid chromatographic analysis.

A new method involving matrix solid-phase dispersion (MSPD) extraction and hydrophilic interaction ultra-high-pressure liquid chromatography (HILIC-UHPLC) with photodiode array detection was developed for the determination of carbadox and olaquindox in feed. Separation of carbadox and olaquindox was achieved within 1 min on the 1.7 microm Acquity UPLC BEH HILIC column by using isocratic elution with a mobile phase consisting of 10 mmol L(-1) ammonium acetate in acetonitrile-water (95:5, v/v) at a flow rate of 0.5 mL min(-1). Optimization of MSPD extraction parameters, such as type of solid sorbent and elution solvent were carried out. Optimal conditions selected for MSPD extraction were: 0.25 g of feed sample, 0.5 g of octadecylsilica as solid sorbent and 10 mL of acetonitrile-methanol (8:2, v/v) as eluting solvent. Both analytes provided average recoveries from spiked feed samples ranging from 89.1 to 98.4% with relative standard deviations less than 10%. Obtained performance characteristics are comparable to those achieved by liquid-liquid extraction-HPLC with the advantages of being simpler and significantly faster.

Validation of an analytical method for the determination of carbadox and olaquindox in feedstuff by liquid chromatography coupled to UV and/or diode array detection.

The performance characteristics of an analytical method based on high-performance liquid chromatography (HPLC) for the detection of the banned growth promoters, carbadox and olaquindox, in feedstuff were determined via a collaborative study. The relative standard deviation of repeatability (RSDr) ranged 1.1-5.5% for carbadox and 2.5-6.2% for olaquindox. The relative standard deviation of reproducibility (RSDR) ranged 6.4-10.7% for carbadox and 12.8-20.0% for olaquindox. In all cases, the HORRAT values were equal or below the critical value of 1.5. Moreover, trueness in all cases was between the acceptance limits of 80 and 110%. Consequently, it was concluded that the method is suitable for quantitative evaluation. The method was also qualitatively assessed in terms of correct identification of the target analytes by examination of the UV spectrum when the more specific diode array detector was coupled to HPLC. In all cases, the percentage of correct identifications was >or=94% for olaquindox and carbadox, while the percentage of false negatives was

Determination of amprolium, carbadox, monensin, and tylosin in surface water by liquid chromatography/tandem mass spectrometry.

Antibiotics present in the environment are recently considered as emerging contaminants, and have raised increasing concerns about their potential risks to ecosystems and human health. In addition to the utilization for treatment, antibiotics are also routinely added as supplements in livestock feed to promote animal growth. A portion of the administered dose used for these purposes can be excreted into animal manure, and land application of the animal manure as plant fertilizers enhances the dissemination of antibiotics in the environment. It is a common practice to simultaneously administer multiple classes of antibiotics to livestock in an animal production farm. This study attempts to develop a protocol to determine four commonly used veterinary pharmaceuticals, amprolium, carbadox, monensin, and tylosin, in surface runoff from a livestock farm. A single-cartridge solid-phase extraction procedure was developed to simultaneously extract these veterinary antibiotics from surface water which were subsequently analyzed by liquid chromatography/tandem mass spectrometry. The extraction recoveries of spiked samples ranged from 89 to 113%, and the limits of quantitation were 8, 25, 1, and 35 ng/L for amprolium, carbodox, monensin, and tylosin, respectively. In the surface runoff from a livestock farm, amprolium was most frequently detected with the concentration range of 10-288 ng/L. Monensin was frequently detected with concentrations up to 37 ng/L. Tylosin was detected in two out of eleven samples, and carbadox was not detected in the surface runoff. The results indicate that the developed analytical method can be utilized to determine multiple classes of veterinary antibiotics present in surface runoff originating from animal farms.

Confirmatory method for the analysis of carbadox and olaquindox in porcine feedingstuffs using LC-electrospray MS-MS.

A method is described for the quantitative determination of the two feed additives carbadox and olaquindox in porcine feedingstuffs. The use of these agents in feedingstuffs was prohibited in the European Union as a result of concerns about their toxicity. Regulatory laboratories are required to have suitably validated analytical methods to ensure compliance with the ban. The analytes were extracted from finished feedingstuffs into acetonitrile:chloroform (1:1, v/v), and aliquots (1.0 ml) of the extract were dried down under a stream of nitrogen at 65 degrees C. All residues were re-dissolved in HPLC mobile phase containing acetonitrile/water/formic acid. Analysis was based on LC coupled to positive-ion electrospray MS-MS, with daughter ions for carbadox at m/z 231 and 90, and for olaquindox at m/z 212 and 143 being monitored. The method was validated by analysing feed samples fortified with carbadox and olaquindox at 0.5, 2.5 and 5 mg kg(-1) on three separate occasions. Sample preparation was simple, thus allowing the confirmation of these compounds in large numbers of samples.

[Determination of carbadox metabolites, quinoxaline-2-carboxylic acid and desoxycarbadox, in swine muscle and liver by liquid chromatography/mass spectrometry].

A sensitive and selective method using liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) for the determination of carbadox metabolites, quinoxaline-2-carboxylic acid (QCA) and desoxycarbadox (Desoxy-CDX), in swine muscle and liver has been developed. The LC separation was performed on a Cadenza CD-C18 column (10 cm x 2 mm i.d.) with a gradient system of 0.01% acetic acid-acetonitrile as the mobile phase at a flow rate of 0.2 mL/min. Negative ionization produced the [M-H]- molecular ion of QCA. On the other hand, the positive mode produced the [M+H]+ ion of Desoxy-CDX. The calibration graphs for QCA and Desoxy-CDX were rectilinear from 0.01 to 0.5 ng with selected ion monitoring (SIM). The drugs were extracted with 0.3% metaphosphoric acid-methanol (7:3), and the extracts were cleaned up on an Oasis HLB cartridge (60 mg) and by liquid-liquid extraction. The recoveries of QCA and Desoxy-CDX from swine muscle and liver fortified at 2.5 and 5 ng/g were 70.2-86.3%, and the detection limits were 1 ng/g for both drugs.

Evaluation of certain veterinary drug residues in food.

The first part of the report presents the views of the Committee on assessment of carcinogenic risk, quality of data, marker residues, and the Joint FAO/WHO Project to update the principles and methods for the risk assessment of chemicals in food. Summaries follow of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: two antimicrobial agents (neomycin and flumequine), an antiprotozoal agent (imidocarb), three insecticides (deltamethrin, dicyclanil, and trichlorfon) and one production aid (carbadox). Annexed to the report is a summary of the Committee's recommendations on these drugs, including Acceptable Daily Intakes and Maximum Residue Limits. Corrigenda to WHO Technical Report Series 911: Evaluation of certain veterinary drug residues in food, 2002, are also included.

High-performance liquid chromatographic determination of carbadox, olaquindox, furazolidone, nitrofurazone, and nitrovin in feed.

A high-performance liquid chromatography with gradient programming method was developed to determine the amount of carbadox (CBX), olaquindox (OLQ), furazolidone (FZ), nitrofurazone (NF), and nitrovin (NTV) in feed simultaneously. Complete separation of the drugs was obtained using a C8 silica gel column with gradients of acetonitrile as mobile phase. The mobile phase used an acetonitrile gradient with an initial hold time of 1 min at 0% acetonitrile, followed by an increase to 50% acetonitrile over 10 min. The correlation coefficients (r) for calibration curves of the five feed additives were greater than 0.999. The relative standard deviations (RSDs) of peak areas from four injections for these drugs at three concentrations were less than 3.0%, although the RSD for NTV at 5 ppm was somewhat large (6.7%). The medicated feeds were extracted by pretreating with water, extracted with 95% dimethylformamide overnight at room temperature, and cleaned up on a column of alumina oxide. Recoveries of CBX, OLQ, FZ, NF, and NTV from low level spiked feed were 102.0, 94.6, 97.4, 110.6, and 66.0%, respectively, and from high level spiked feed, they were 114.09, 99.1, 97.3, 109.9, and 62.7%, respectively.

Column liquid chromatographic determination of carbadox and olaquindox in feeds.

A column liquid chromatographic method for simultaneous determination of carbadox and olaquindox in swine feeds is described. The drugs were extracted from feeds with carbon tetrachloride-dimethylformamide (80:20) at 60 degrees C for 30 min. The extract was mixed with water (25:45). After centrifugation the aqueous layer was chromatographed on a reversed-phase column using gradient elution and ultraviolet detection at wavelengths of 305 and 262 nm. Recoveries from samples fortified at levels of 20-50 ppm were 92 +/- 9% for carbadox and 93 +/- 6% for olaquindox (means +/- standard deviations, n = 71).

Determination of carbadox-related residues in swine liver by gas chromatography/mass spectrometry with ion trap detection.

The ion trap detector (ITD), in combination with a capillary gas chromatograph and under chemical ionization conditions, offers sufficient sensitivity to determine carbadox-related residues as the methyl ester derivative of quinoxaline-2-carboxylic acid at 3 micrograms/kg or higher in porcine liver. A tetradeuterated internal standard of QME effectively compensates for losses incurred during sample preparation. The method produced mean levels of 3.3 (+/- 0.5), 5.5 (+/- 0.8), and 10.1 (+/- 0.9) micrograms/kg for liver fortified at 3, 5, and 10 micrograms/kg. When applied to analysis of samples containing incurred residues of 14C-carbadox at the low microgram/kg level, results were comparable to those obtained by reverse isotope dilution analysis.

Optimization and ruggedness testing of the determination of residues of carbadox and metabolites in products of animal origin. Stability studies in animal tissues.

A method developed for the determination of residues of carbadox and its metabolites in swine tissues using high-performance liquid chromatography with on-line precolumn enrichment and postcolumn derivatization with UV-VIS detection was optimized and the applicability of the method was extended to plasma and eggs. With the optimized method, more than twenty samples per person per day can be analysed. In the matrices investigated, the observed limit of determination for carbadox is 0.5-1 micrograms/kg and for desoxy-carbadox 0.5-2 micrograms/kg. The mean recovery for desoxy-carbadox in kidney, muscle and liver as established by two laboratories over a 2-month period is 95% (relative standard deviation = 14%, N = 37, 10 micrograms/kg). In other matrices the recoveries are between 83 and 91%. The recovery for carbadox is 70-80% in muscle, plasma and eggs. The method has been used routinely in pharmacokinetic and surveillance studies. Stability studies of kidney and liver samples spiked with carbadox showed that carbadox is rapidly decomposed (in vitro metabolism). After storage for about 1 h at 4 degrees C, more than 50% of the added amount is converted by reduction to desoxy-carbadox. In contrast, carbadox is stable in eggs and muscle under spiking conditions and during storage at -20 degrees C. Desoxy-carbadox is stable during spiking and storage at -20 degrees C in eggs and muscle. In kidney and liver, its stability was good under spiking conditions but could not be proved unequivocally during storage.