Quinoxaline-2-carboxylic Acid Research Articles

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.

Determination of 3-Methyl-quinoxaline-2-carboxylic Acid and Quinoxaline-2-carboxylic Acid in Pork Based on a Background Fluorescence Quenching Immunochromatographic Assay

A novel rapid method based on a background fluorescence quenching immunochromatographic assay (bFQICA) was established to achieve simultaneously the quantitative detection of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and quinoxaline-2-carboxylic acid (QCA), which were efficiently extracted and enriched 4 times using immunomagnetic beads from pork. The analysis of field pork samples by bFQICA was in accordance with that of LC-MS/MS; especially, the proposed bFQICA exhibited great advantages in convenience and efficiency, which only takes 30 min for the detection of MQCA and QCA.

Development of a Sensitive Chemiluminescent Immunoassay for the Determination of 3-Methyl-quinoxaline-2-carboxylic Acid and Quinoxaline-2-carboxylic Acid in Edible Animal Tissues Using Immunomagnetic Beads Capturing.

We have reported a sensitive chemiluminescent competitive indirect enzyme-linked immunosorbent assay (CL-ciELISA) based on immunomagnetic beads separation, purification and enrichment for the simultaneous determination of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and quinoxaline-2-carboxylic acid (QCA) in edible animal tissues. Forty field samples were analyzed with the developed CL-ciELISA, and the results correlated well with those obtained in liquid chromatography-tandem mass spectrometry (LC-MS/MS), confirming the utility of CL-ciELISA for the quantitation of MQCA and QCA in fish, shrimp, pork and chicken with a good accuracy.

Luminescent Molecularly Imprinted Polymers Based on Covalent Organic Frameworks and Quantum Dots with Strong Optical Response to Quinoxaline-2-Carboxylicacid.

Three-dimensional molecularly imprinted polymers (MIPs) based on quantum dots-grafted covalent organic frameworks (QDs-grafted COFs) are reported in this study. The compound 1,3,5-triformylphloroglucinol-P-phenylenediamine was used as COF material to react with the amino-modified CdSe/ZnS QDs by Schiff-base reactions. The amino-derived QDs reacted with quinoxaline-2-carboxylicacid (QCA) via a non-covalent interaction. The system combines the advantages of MIPs, COFs, and QDs for highly sensitive and selective QCA detection. The MIPs based on QDs-grafted COFs showed good chemical selectivity and thermal stability, as well as consistency in QCA optosensing. Under optimal conditions, the detection limit for QCA in meat and feed samples was 0.85 μmol L−1, over a linear concentration range of 1–50 μmol L−1. The current findings suggest a potential application of MIPs based on QDs-grafted COFs for the detection of trace levels of hazardous chemicals for food safety and environmental control.

A Convenient and Sensitive LC-MS/MS Method for Simultaneous Determination of Carbadox- and Olaquindox-Related Residues in Swine Muscle and Liver Tissues

This paper presents a convenient and sensitive LC-MS/MS method for the simultaneous determination of carbadox and olaquindox residues, including desoxyolaquindox (DOLQ), desoxycarbadox (DCBX), quinoxaline-2-carboxylic acid (QCA), 3-methyl-quinoxaline-2-carboxylic acid (MQCA), and the glycine conjugates of QCA and MQCA (namely, QCA-glycine and MQCA-glycine, resp.) in swine muscle and liver tissues. Tissue samples were extracted with 2% metaphosphoric acid in 20% methanol and cleaned up by solid-phase extraction (SPE) on a mixed-mode anion-exchange column (Oasis MAX). Analysis was performed on a C18 column by detection with mass spectrometry in the multiple reaction monitoring (MRM) mode. The limits of detection (LODs) of the six analytes were determined to be 0.01 μg·kg−1 to 0.25 μg·kg−1, and the limits of quantification (LOQs) were 0.02 μg·kg−1 to 0.5 μg·kg−1. The total recoveries of the six analytes in all tissues were higher than 79.1% with the RSD% less than 9.2%. The developed method can determine the real residue level of QCA and MQCA, whether they are present in free form or as glycine conjugates in tissues, together with the carcinogenic desoxy metabolites DCBX and DOLQ with high recovery. Therefore, this method was suitable for routine analysis of residue control programmes and the residue depletion study of CBX and OLQ on swine.

Detecting quinoxaline-2-carboxylic acid in animal tissues by using sensitive rapid enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluoroimmunoassay (TR-FIA) based on an anti-N-butylquinoxaline-2-carboxamide (BQCA) monoclonal antibody were standardized and validated for quinoxaline-2-carboxylic acid (QCA) screening in animal tissues and its performance were compared to HPLC. The sensitivities obtained for edible tissue extracts were 1.62 and 1.12ngml−1 for ic-ELISA and TR-FIA detection, respectively. Two samples were spiked with QCA and analyzed by both methods. The recovery values ranged from 92.6% to 112.2% and the coefficients of variation were less than 15% for QCA spiking into swine tissue samples at concentrations of 2.5–50.0μgkg−1. Excellent correlations (r2=0.987–0.996) of the ic-ELISA/HPLC and TR-FIA/HPLC data were observed for processed samples. The results demonstrated that the ic-ELISA and TR-FIA methods were rapid and accurate for the residue detection of QCA in animal tissues.

A physiologically based pharmacokinetic model for quinoxaline-2-carboxylic acid in rats, extrapolation to pigs.

A multi-compartment physiologically based pharmacokinetic (PBPK) model to describe the disposition of cyadox (CYX) and its metabolite quinoxaline-2-carboxylic acid (QCA) after a single oral administration was developed in rats (200 mg/kg b.w. of CYX). Considering interspecies differences in physiology and physiochemistry, the model efficiency was validated by pharmacokinetic data set in swine. The model included six compartments that were blood, muscle, liver, kidney, adipose, and a combined compartment for the rest of tissues. The model was parameterized using rat plasma and tissue concentration data that were generated from this study. Model simulations were achieved using a commercially available software program (ACSLXL ibero version 3.0.2.1). Results supported the validity of the model with simulated tissue concentrations within the range of the observations. The correlation coefficients of the predicted and experimentally determined values for plasma, liver, kidney, adipose, and muscles in rats were 0.98, 0.98, 0.98, 0.99, and 0.95, respectively. The rat model parameters were then extrapolated to pigs to estimate QCA disposition in tissues and validated by tissue concentration of QCA in swine. The correlation coefficients between the predicted and observed values were over 0.90. This model could provide a foundation for developing more reliable pig models once more data are available.

Sensitive and selective electrochemical determination of quinoxaline-2-carboxylic acid based on bilayer of novel poly(pyrrole) functional composite using one-step electro-polymerization and molecularly imprinted poly(o-phenylenediamine)

A facile and efficient molecularly imprinted polymer (MIP) recognition element of electrochemical sensor was fabricated by directly electro-polymerizing monomer o-phenylenediamine (oPD) in the presence of template quinoxaline-2-carboxylic acid (QCA), based on one-step controllable electrochemical modification of poly(pyrrole)-graphene oxide-binuclear phthalocyanine cobalt (II) sulphonate (PPY-GO-BiCoPc) functional composite on glassy carbon electrode (GCE). The MIP film coated on PPY-GO-BiCoPc functional composite decorated GCE (MIP/PPY-GO-BiCoPc/GCE) was presented for the first time. The synergistic effect and electro-catalytic activity toward QCA redox of PPY-GO-BiCoPc functional composite were discussed using various contrast tests. Also, the effect of experimental variables on the current response such as, electro-polymerization cycles, template/monomer ratio, elution condition for template removal, pH of the supporting electrolyte and accumulation time, were investigated in detail. Under the optimized conditions, the proposed MIP sensor possessed a fast rebinding dynamics and an excellent recognition capacity to QCA, while the anodic current response of square wave voltammetry (SWV) was well-proportional to the concentration of QCA in the range of 1.0×10−8–1.0×10−4 and 1.0×10−4–5.0×10−4molL−1 with a low detection limit of 2.1nmolL−1. The established sensor was applied successfully to determine QCA in commercial pork and chicken muscle samples with acceptable recoveries (91.6–98.2%) and satisfactory precision (1.9–3.5% of SD), demonstrating a promising feature for applying the MIP sensor to the measurement of QCA in real samples.

Tissue Deposition and Residue Depletion of Cyadox and Its Three Major Metabolites in Pigs after Oral Administration

Tissue deposition and residue depletion profiles of cyadox (Cyx) and its three major metabolites, including 1,4-bisdesoxycyadox (Cy1), 4-desoxycyadox (Cy2), and quinoxaline-2-carboxylic acid (QCA), in pigs after multiple oral administrations were determined. Thirty-five healthy adult pigs were randomly divided into seven groups and orally treated with Cyx at a dosage of 20 mg/kg of body weight for five consecutive days. Each group of five pigs was randomly slaughtered 12, 24, 72, 120, 168, 216, and 264 h after the last dosing, and tissue samples, including muscle, liver, kidney, and fat, were collected and analyzed via the liquid chromatography-tandem mass spectrometry method. The concentration-time data of Cyx and its three metabolites (Cy1, Cy2, and QCA) were analyzed with WinNonlin. Results showed that metabolites of Cyx were quickly generated in swine tissues and the concentrations of QCA in kidney were higher than those of Cyx and other metabolites in all edible tissues. These results provide further insight into the metabolism of Cyx and confirmation of the residue marker and target tissue of Cyx in pigs.

Electrochemical sensor based on molecularly imprinted polymer film via sol–gel technology and multi-walled carbon nanotubes-chitosan functional layer for sensitive determination of quinoxaline-2-carboxylic acid

Quinoxaline-2-carboxylic acid (QCA) is difficult to measure since only trace levels are present in commercial meat products. In this study, a rapid, sensitive and selective molecularly imprinted electrochemical sensor for QCA determination was successfully constructed by combination of a novel modified glassy carbon electrode (GCE) and differential pulse voltammetry (DPV). The GCE was fabricated via stepwise modification of multi-walled carbon nanotubes (MWNTs)-chitosan (CS) functional composite and a sol–gel molecularly imprinted polymer (MIP) film on the surface. MWNTs-CS composite was used to enhance the electron transfer rate and expand electrode surface area, and consequently amplify QCA reduction electrochemical response. The imprinted mechanism and experimental parameters affecting the performance of MIP film were discussed in detail. The resulting MIP/sol–gel/MWNTs-CS/GCE was characterized using various electrochemical methods involving cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and DPV. The sensor using MIP/sol–gel/MWNTs-CS/GCE as working electrode showed a linear current response to the target QCA concentration in the wide range from 2.0×10−6 to 1.0×10−3molL−1 with a low detection limit of 4.4×10−7molL−1 (S/N=3). The established sensor with excellent reproductivity and stability was applied to evaluate commercial pork products. At five concentration levels, the recoveries and standard deviations were calculated as 93.5–98.6% and 1.7–3.3%, respectively, suggesting the proposed sensor is promising for the accurate quantification of QCA at trace levels in meat samples.

Pharmacokinetics of Cyadox and Its Major Metabolites in Swine After Intravenous and Oral Administration

Pharmacokinetics of cyadox (CYX) and its major metabolites in healthy swine was investigated in this paper. 1,4-Bisdesoxycyadox (BDCYX), cyadox-1-monoxide (CYX-1-O) and quinoxaline-2-carboxylic acid (QCA), three main metabolites of cyadox, were synthesized by College of Science, China Agricultural University. Cyadox (CYX) was administered to 8 healthy cross-bread swine intravenously (i.v.) and orally (p. o.) at a dosage of 1 mg kg−1 body weight and 40 mg kg−1 body weight respectively in a randomized crossover design test with 2-wk washout period. A sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the determination of cyadox and its major metabolites in plasma. CYX and its major metabolites BDCYX, and CYX-1-O can be detected after intravenous administration of cyadox while CYX and its metabolites BDCYX, CYX-1-O and QCA can be detected after oral administration of CYX. Plasma concentration vs. time profiles of CYX and its major metabolites were analyzed by non-compartmental pharmacokinetic method. Following i.v. administration, the areas under the plasma concentration-time curve (AUC0-∞) were (0.38±0.03) μg mL−1 h (CYX), (0.018±0.002) μg mL−1 h (BDCYX) and (0.17±0.02) μg mL−1 h (CYX-1-O), respectively. The terminal elimination half-lives (t1/2lz) were determined to be (0.93±0.07) h (CYX), (1.45±0.04) h (BDCYX), and (0.92±0.04) h (CYX-1-O), respectively. Steady-state distribution volume (Vss) of (2.14±0.11) L kg−1 and total body clearance (CL) of (2.84±0.19) L h−1 kg−1 were determined for CYX after i.v. dosing. The bioavailability (F) of CYX was 2.85% for oral administration. After single i.v. administration, peak plasma concentrations (Cmax) of (1.08±0.06) μg mL−1 (CYX), (0.0068± 0.0004) μg mL−1 (BDCYX) and (0.25±0.03) μg mL−1 (CYX-1-O) were observed at Tmax of 0.033 h (CYX), 1 h (BDCYX) and 0.033 h (CYX-1-O), respectively. The main pharmacokinetic parameters after p.o. administration were as follows: AUC0-∞ were (0.42±0.04) μg mL−1 h (CYX), (1.38±0.14) μg mL−1 h (BDCYX), (0.59±0.02) μg mL−1 h (CYX-1-O) and (1.48±0.09) μg mL−1 h (QCA), respectively. t1/2lz were (4.77±0.33) h (CYX), (5.77±0.56) h (BDCYX), (4.12±0.28) h (CYX-1-O), and (8.51±0.39) h (QCA), respectively. After p.o. administration, Cmaxs of (0.033±0.002) μg mL−1 (CYX), (0.22±0.03) μg mL−1 (BDCYX), (0.089±0.005) μg mL−1 (CYX-1-O), and (0.17± 0.01) μg mL−1 (QCA) were observed at Tmax of (7.38±0.33) h (CYX), (7.25±0.31) h (BDCYX), (7.38±0.33) h (CYX-1-O), and (7.25±0.31) h (QCA), respectively. The results showed that CYX was slowly absorbed after oral administration and most of CYX was transformed to its metabolites in swine. The area under plasma concentration-time curve (AUC0-∞) of metabolites were higher than that of CYX after p.o. administration, and the elimination half-lives (t1/2lz) of QCA were longer than those of CYX, CYX-1-O, and BDCYX after oral administration.

In Vitro Characterization of Echinomycin Biosynthesis: Formation and Hydroxylation of L-Tryptophanyl-S-Enzyme and Oxidation of (2S,3S) β-Hydroxytryptophan

Quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA) feature in quinomycin family and confer anticancer activity. In light of the significant potency against cancer, the biosynthetic gene clusters have been reported from many different Streptomyces strains, and the biosynthetic pathway were proposed mainly based on the in vivo feeding experiment with isotope labeled putative intermediates. Herein we report another gene cluster from Streptomyces griseovariabilis subsp. bandungensis subsp. nov responsible for the biosynthesis of echinomycin (a member of quinomycin family, also named quinomycin A) and presented in vitro evidence to corroborate the previous hypothesis on QXC biosynthesis, showing that only with the assistance of a MbtH-like protein Qui5, did the didomain NRPS protein (Qui18) perform the loading of a L-tryptophan onto its own PCP domain. Particularly, it was found that Qui5 and Qui18 subunits form a functional tetramer through size exclusion chromatography. The subsequent hydroxylation on β-carbon of the loaded L-tryptophan proved in vitro to be completed by cytochrome P450-dependent hydroxylase Qui15. Importantly, only the Qui18 loaded L-tryptophan can be hydroxylated by Qui15 and the enzyme was inactive on free L-tryptophan. Additionally, the chemically synthesized (2S,3S) β-hydroxytryptophan was detected to be converted by the tryptophan 2,3-dioxygenase Qui17 through LC-MS, which enriched our previous knowledge that tryptophan 2,3-dioxygenase nearly exclusively acted on L-tryptophan and 6-fluoro-tryptophan.

A sensitive and selective imprinted solid phase extraction coupled to HPLC for simultaneous detection of trace quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid in animal muscles

A new molecularly imprinted polymer (MIP), selective for major metabolites of quinoxaline-1,4-dioxides, was prepared through bulk polymerisation using quinoxaline-2-carboxylic acid (QCA) as template, diethylaminoethylmethacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker in tetrahydrofuran. The synthesised MIP was characterised by Fourier transform infrared and adsorption experiments. MIP exhibited high affinity, fast kinetics for QCA and good selectivity for QCA and methyl-3-quinoxaline-2-carboxylic acid (MQCA). MIP obtained was used as a selective sorbent for molecularly imprinted solid phase extraction (MISPE) coupled with HPLC to detect QCA and MQCA. Under the optimal conditions, the limits of detection (S/N=3) of porcine, chicken and fish muscles were 0.1, 0.3, 0.1μg/kg for QCA and 0.2, 0.3, 0.1μg/kg for MQCA, respectively and good recoveries were obtained in the range from 60.0 to 119.4%. These results indicated the MISPE–HPLC procedure could be successfully used for the determination QCA and MQCA in animal muscles.

Determination of the residues of 3-methyl-quinoxaline-2-carboxylic acid and quinoxaline-2-carboxylic acid in animal origin foods by high performance liquid chromatography-tandem mass spectrometry

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) method was developed for the simultaneous quantitative determination of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and quinoxaline-2-carboxylic acid (QCA) as the marker residues for carbadox (CBX) and olaquindox (OLA), respectively, in the muscles and livers of porcine and chicken and in the muscles of fish and shrimp. The MQCA and QCA were deproteinated with 5% metaphosphoric acid in 10% methanol followed by liquid-liquid extraction. Further clean-up was performed by solid phase extraction (SPE) through mixed mode anion-exchange columns (Oasis MAX SPE). The separation of the compounds was carried on a Waters Xterra MS C18 column (150 mm x 2.1 mm, 5 microm) by a gradient elution using methanol and 0.2% formic acid as mobile phases. The analytes were detected by tandem mass spectrometry in multiple reaction monitoring (MRM) mode with positive electrospray ionization. The MQCA and QCA were quantified by internal standard method. The linear ranges were 1.0-20.0 microg/L and the correlation coefficients were not less than 0.9996. The average recoveries and relative standard deviations ranged from 62.4%-118% and 1.48%-28.1% respectively at the spiked levels of 0.1, 0.2 and 1.0 microg/kg for the both markers. The limit of quantitation (LOQ) was 0.1 microg/kg. The method is sensitive, accurate and suitable for the determination and confirmation of MQCA and QCA in animal origin foods.

A specific UPLC-ESI-MS/MS method for analysis of cyadox and its three main metabolites in fish samples

A rapid and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of cyadox (CYX) and its three major metabolites, quinoxaline-2-carboxylic acid (QCA), 1-desoxycyadox (1-DCYX) and 1,4-bisdesoxycyadox (BDCYX) in fish. Samples were extracted with acetonitrile–0.5 M hydrochloric acid (9 + 1) and cleaned up using Oasis MAX cartridges. The total time taken for separation by UPLC was less than 5 min. The four target compounds were then determined by ESI-MS/MS. At the fortified levels of 2–50 μg kg−1 in fish, recoveries of all the compounds ranged from 80.2% to 88.2%, with a relative standard deviation of 6.8 to 14.6%. Limits of detection for CYX, QCA, 1–DCYX and BDCYX were 0.52, 1.07, 0.69 and 0.38 μg kg−1, respectively.

Novel surface molecularly imprinted sol–gel polymer applied to the online solid phase extraction of methyl-3-quinoxaline-2-carboxylic acid and quinoxaline-2-carboxylic acid from pork muscle

A new molecularly imprinted polymer (MIP), selective for major metabolites of quinoxaline-1,4-dioxides was firstly prepared by combining surface molecular imprinting technique with the sol-gel process. Methyl-3-quinoxaline-2-carboxylic acid (MQCA) was used as template, 3-aminopropyltriethoxysilane as functional monomer, and tetraethoxysilicane as cross-linker. The MIP was characterized by Fourier transform infrared and evaluated through static adsorption experiments. The results indicated that MIP had high adsorption capacity, fast binding kinetics for MQCA, and the polymer showed a high degree of cross-reactivity for quinoxaline-2-carboxylic acid (QCA). The MIP was then applied as a selective sorbent in an online solid phase extraction (SPE) coupled with high-performance liquid chromatography (HPLC). For a 50-mL sample solution, enrichment factors of 1,349 and 1,046 for QCA and MQCA, respectively, and limits of detection (S/N=3) of 0.8 and 2 ng L(-1) for QCA and MQCA, respectively, were obtained (corresponding to 0.02 and 0.04 ng g(-1) in solid samples for final 100 mL of sample solutions of 5 g of pork). The sample preparation protocol was simplified and only included one step extraction with acetonitrile (MeCN) after the release of target analytes through acidic hydrolysis without further sample cleanup. The new MIP-SPE-HPLC method was successfully applied to the quantification of trace QCA and MQCA in pork muscle with good recoveries ranging from 67% to 80% and RSD below 8%.

Involvement of common intermediate 3-hydroxy-L-kynurenine in chromophore biosynthesis of quinomycin family antibiotics

Quinomycin antibiotics, represented by echinomycin, are an important class of antitumor antibiotics. We have recently succeeded in identification of biosynthetic gene clusters of echinomycin and SW-163D, and have achieved heterologous production of echinomycin in Escherichia coli. In addition, we have engineered echinomycin non-ribosomal peptide synthetase to generate echinomycin derivatives. However, the biosynthetic pathways of intercalative chromophores quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA), which are important for biological activity, were not fully elucidated. Here, we report experiments involving incorporation of a putative advanced precursor, (2S, 3R)-[6'-(2)H]-3-hydroxy-L-kynurenine, and functional analysis of the enzymes Swb1 and Swb2 responsible for late-stage biosynthesis of HQA. On the basis of these experimental results, we propose biosynthetic pathways for both QXC and HQA through the common intermediate 3-hydroxy-L-kynurenine.

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 5 μg kg −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 ng kg −1 and QCA and mQCA residues at concentrations ≥0.50 ng kg −1in bovine muscle, pork liver and muscle tissues.

Development of high performance liquid chromatographic methods for the determination of cyadox and its metabolites in plasma and tissues of chicken

Cyadox (CYX) is an antimicrobial growth-promoter of the quinoxalines. It is highly effective on improving growth and feed conversion of chicken with little toxicity. For food safety concerns, HPLC-UV methods were developed for the sequential determination of CYX and its major metabolites, 1,4-bisdesoxycyadox (BDCYX) and quinoxaline-2-carboxylic acid (QCA), in plasma, muscle, liver, kidney and fat of chicken. For CYX and BDCYX, samples were subjected to a deproteinisation, a degrease and a liquid–liquid extraction. For QCA, samples were subjected to an alkali hydrolysis, a liquid–liquid extraction and a cation exchange column (AG MP-50 resin) clean-up. Analysis was performed on a RP-C18 column by UV detection with a gradient program of wavelength. Gradient elution was performed at a flow of 1 mL/min. The limits of quantification for CYX, BDCYX and QCA in plasma and tissues were 0.025 μg/g, and 0.002 μg/g for QCA in muscle. The recoveries of three compounds in plasma and tissues were 70–87% with inter-day relative standard deviation (R.S.D.) less than 10%. An animal experiment was performed to show the applicability of the present methods in real biological samples, which demonstrated a satisfactory applicability since all compounds could be detected nearly in all tissues. The present methods were highly sensitive and accurate, and could therefore be useful in pharmacokinetic and residue studies for cyadox in chicken. The developed methods will be further applied in the residue screening of cyadox and its metabolites in chicken.

Improved Production of Triostin A in Engineered Escherichia coli with Furnished Quinoxaline Chromophore by Design of Experiments in Small-Scale Culture

Proficient production of the antitumor agent triostin A was developed using engineered Escherichia coli (E. coli). The bacterium played host to 15 genes that encode integral biosynthetic proteins which were identified and cloned from Streptomyces lasaliensis. In this study, triostin A production was dramatically increased by more than 20-fold, 13 mg/L, with the introduction of exogenous quinoxaline-2-carboxylic acid (QXC), the speculative starting unit for biosynthesis of triostin A. Conversely, de novo production of triostin A by means of high cell density fed-batch fermentation that is exclusive of exogenous QXC bore a modest amount of the antitumor agent. Noteworthy production of the biologically active molecule was achieved with small-scale cultivation and quantitative analysis of the product was accomplished with a liquid chromatography-mass spectrometer. This simple and speedy system could easily provide us with valuable information for maximizing the production titer. Our entirely heterologous production system also establishes a basis for the future use of E. coli for generation of novel bioactive compounds through tolerable precursor-directed biosynthesis.