Quinoxaline-2-carboxylic Acid Research Articles

Development of a high-performance liquid chromatography method for the simultaneous quantification of quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid in animal tissues

A method of high-performance liquid chromatography with UV detection has been established for simultaneous quantitative determination of quinoxaline-2-carboxylic acid (QCA) and methyl-3-quinoxaline-2-carboxylic acid (MQCA), the marker residues for carbadox (CBX) and olaquindox (OLA), respectively, in the muscles and livers of porcine and chicken and in the muscle of fish. Tissue samples were subject to acid hydrolysis followed by liquid–liquid extraction and Oasis MAX solid-phase extraction clean-up. The method was validated according to the EU Commission Decision 2002/657/EC. The decision limits (CCα) were 0.7–2.6 μg/kg and the detection capabilities (CCβ) were 1.3–5.6 μg/kg for QCA and MQCA in tissues. The recoveries of QCA and MQCA, spiked at levels of 2–100 μg/kg, were from 70 to 110%; the relative standard deviation values were <20%. This simple, fast and economic method could be applied to the monitoring for the possible misuse of CBX and OLA in animal edible tissue samples.

Identification and Stereochemical Assignment of the β-Hydroxytryptophan Intermediate in the Echinomycin Biosynthetic Pathway

[reaction: see text] Little is known about how quinoxaline-2-carboxylic acid (QC) is synthesized in nature. On the basis of analysis of echinomycin biosynthetic gene clusters as well as feeding experiments with labeled precursors, we have proposed a biosynthetic pathway to QC and identified the (2S,3S)-beta-hydroxytryptophan as a key intermediate.

Development of HPLC Methods for the Determination of Cyadox and Its Main Metabolites in Goat Tissues

Rapid and sensitive high-performance liquid-chromatographic methods were developed for the determination of cyadox, an antimicrobial growth-promoter, and its main metabolites (1,4-bisdesoxycyadox, quinoxaline-2-carboxylic acid) in goat muscle, liver, kidney and fat. Cyadox (CYX) and 1,4-bisdesoxycyadox (BDCYX) in fat were extracted with acetonitrile, and in other tissues with ethyl acetate. Quinoxaline-2-carboxylic acid (QCA) was isolated from tissue hydrolysates by solvent extraction, cleaned up with ion-exchange chromatography, followed by a final liquid-liquid extraction step. UV detections of CYX, BDCYX and QCA were performed at 305, 280 and 320 nm, respectively. The average recoveries of CYX, BDCYX and QCA in spiked tissues at levels of 25, 50, 100 microg/kg were 65 - 92%. The inter-day relative standard deviation for three compounds in different tissues was 5 - 16%. The quantitation limit was 25 microg/kg, and the detection limit was 15 microg/kg for three compounds in various tissues. Incurred goat tissues were analyzed to demonstrate the validity of the described methodologies. The present methods were highly selective and could be used in the metabolism and residue studies of cyadox.

Effects of Cooking and Storage on Residues of Cyadox in Chicken Muscle

The aim of this study was to investigate the depletion of residues of cyadox in chicken muscle over time. The heat stabilities of cyadox (CYX) and its two metabolites, 1,4-bisdesoxycyadox (BDCYX) and quinoxaline-2-carboxylic acid (QCA), in water, cooking oil, and as incurred residues in chicken muscle were investigated. CYX was shown to be unstable with a half-life of about 37.7 min in 100 degrees C water. In hot cooking oil at 180 degrees C, all three compounds were unstable. CYX decreased quickly and was not able to be detected after heating for 2 min. Diode-array analysis of CYX standard solution in cooking oil indicated that a portion of BDCYX was formed. The residues of CYX and BDCYX deteriorated rapidly in frozen storage, while that of QCA changed slowly. Muscles containing CYX residues were boiled, microwaved, or fried for the specified times. During boiling, CYX and BDCYX were reduced 94% and 81% in 10 min, respectively. During microwave cooking, CYX and BDCYX were reduced 54% and 47% in 2.5 min, respectively. During frying, CYX and BDCYX were reduced 86% and 76%, respectively. No significant reduction of QCA was found for the three cooking methods. The half-lives of CYX residues in cooked chicken muscles were estimated as follows: 2.22 min for CYX and 4.44 min for BDCYX by boiling; 6.66 min for CYX and 9.36 min for BDCYX by microwaving.

Functional Cross-talk between Fatty Acid Synthesis and Nonribosomal Peptide Synthesis in Quinoxaline Antibiotic-producing Streptomycetes

Quinoxaline antibiotics are chromopeptide lactones embracing the two families of triostins and quinomycins, each having characteristic sulfur-containing cross-bridges. Interest in these compounds stems from their antineoplastic activities and their specific binding to DNA via bifunctional intercalation of the twin chromophores represented by quinoxaline-2-carboxylic acid (QA). Enzymatic analysis of triostin A-producing Streptomyces triostinicus and quinomycin A-producing Streptomyces echinatus revealed four nonribosomal peptide synthetase modules for the assembly of the quinoxalinoyl tetrapeptide backbone of the quinoxaline antibiotics. The modules were contained in three protein fractions, referred to as triostin synthetases (TrsII, III, and IV). TrsII is a 245-kDa bimodular nonribosomal peptide synthetase activating as thioesters for both serine and alanine, the first two amino acids of the quinoxalinoyl tetrapeptide chain. TrsIII, represented by a protein of 250 kDa, activates cysteine as a thioester. TrsIV, an unstable protein of apparent Mr about 280,000, was identified by its ability to activate and N-methylate valine, the last amino acid. QA, the chromophore, was shown to be recruited by a free-standing adenylation domain, TrsI, in conjunction with a QA-binding protein, AcpPSE. Cloning of the gene for the QA-binding protein revealed that it is the fatty acyl carrier protein, AcpPSE, of the fatty acid synthase of S. echinatus and S. triostinicus. Analysis of the acylation reaction of AcpPSE by TrsI along with other A-domains and the aroyl carrier protein AcmACP from actinomycin biosynthesis revealed a specific requirement for AcpPSE in the activation and also in the condensation of QA with serine in the initiation step of QA tetrapeptide assembly on TrsII. These data show for the first time a functional interaction between nonribosomal peptide synthesis and fatty acid synthesis.

Confirmation of carbadox and olaquindox metabolites in porcine liver using liquid chromatography–electrospray, tandem mass spectrometry

A method is described for the quantitative determination of quinoxaline-2-carboxylic acid (QCA) and methyl-3-quinoxaline-2-carboxylic acid (MQCA), the metabolites that have been designated as the marker residues for the veterinary drugs, carbadox and olaquindox, respectively, in swine tissue. The method is suitable for use as a confirmatory method under EU National Surveillance Schemes. Porcine liver samples were subjected to protease digestion followed by liquid–liquid extraction. Further clean-up was performed by automated solid phase extraction (SPE) and was followed by a final liquid–liquid extraction step. Analysis was performed using a narrow bore column HPLC coupled to electrospray MS/MS, operated in positive ion mode. MS/MS product ions were monitored at m/ z 102 and 75 amu for QCA, m/ z 145 and 102 amu for MQCA and at m/ z 106 and 152 amu for the d 4-QCA and d 7-MQCA internal standards, respectively. The method has been validated at 3.0, 10, 50 and 150 μg kg −1 for both metabolites. The method performance characteristics—the decision limit (CC α) and the detection capability (CC β) have been determined for QCA at 0.4 and 1.2 μg kg −1, respectively, and for MQCA at 0.7 and 3.6 μg kg −1, respectively.

Quinoxaline-2-carboxylic acid in pigs: criteria to distinguish between the illegal use of carbadox and environmental contamination

Carbadox cannot be used in food-producing animals within the European Union following the adoption of Commission Regulation EC 2788/98/EC. Monitoring of the longest remaining residue—quinoxaline-2-carboxylic acid (QCA)—is the most effective way of enforcing the prohibition on its use. The study was under taken to determine if QCA could be passed from pig to pig following the exposure of unmedicated animals to housing that had previously contained medicated animals. Drug-withdrawal studies were also carried out on medicated animals. Distinction between treated animals and those exposed to QCA might be required by competent national authorities to determine whether a positive result for QCA in tissue is truly ‘violative’. Comparison of the ratio concentrations of QCA in tissues and body fluids was made to determine if they could be used as criteria for discrimination between illegally treated animals and environmental contamination.

Determination of quinoxaline-2-carboxylic acid, the major metabolite of carbadox, in porcine liver by isotope dilution gas chromatography–electron capture negative ionization mass spectrometry

A sensitive and robust method using isotope dilution gas chromatography–electron capture negative chemical ionization mass spectrometry (GC–ECNI-MS) was developed and validated for the analysis of quinoxaline-2-carboxylic acid (QCA) in porcine liver. [ 2H 4]QCA was added to liver samples which were then deproteinated with 2% metaphosphoric acid in 20% methanol. Followed by sequential extraction with water-saturated ethyl acetate and phosphate buffer, the buffer extracts were subject to solid-phase extraction clean-up by mixed mode anion-exchange columns. QCA was derivatized with N-methyl- N- tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) prior to GC–ECNI-MS determination. For unambiguous identification, a second GC–ECNI-MS experiment was performed on suspected positive samples which were derivatized independently with another derivatization agent, trimethylsilyldiazomethane. Excellent recovery and precision were obtained and the limit of quantitation was 0.7 μg/kg (S/N>60). Method ruggedness by Taguchi orthogonal array technique is also presented.

ＬＣ／ＭＳによる豚筋肉および肝臓中のカルバドックス代謝体（キノキサリン‐２‐カルボン酸およびデスオキシカルバドックス）の定量

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.

Development and validation of an improved method for confirmation of the carbadox metabolite, quinoxaline-2-carboxylic acid, in porcine liver using LC-electrospray MS-MS according to revised EU criteria for veterinary drug residue analysis.

A method is described for the quantitative determination of quinoxaline-2-carboxylic acid (QCA), the marker residue for the veterinary drug carbadox, in swine liver. Tissue is subjected to alkaline hydrolysis followed by liquid-liquid extraction. QCA residues are cleaned up using automated solid phase extraction (SPE), before a final liquid-liquid extraction step. Analysis is based on LC coupled to positive ion electrospray MS-MS, monitoring product ions at m/z 129, 102 and 75 for QCA and at m/z 106 for the internal standard (d4-QCA). The method has been validated according to draft revised EU criteria for analysis of veterinary drug residues, and is suitable for monitoring tissues taken under national surveillance schemes. The method has been validated at 3, 10, 30, 100 and 300 microg kg(-1). The method performance characteristics, CCalpha (decision limit) and CCbeta (detection limit) were determined to be 0.16 and 0.27 microg kg(-1), respectively. The described method, which is relatively rapid and applicable to large sample numbers, correlates well (r2 = 0.9799) with a widely used GC-MS assay for QCA.

Quinoxaline-2-carboxylic acid (QCA) in swine liver and muscle.

The concentration of quinoxaline-2-carboxylic acid (QCA) determined by HPLC after alkaline hydrolysis of liver and muscle of swine, ranged from < 3 ng/g to 45.3 ng/g in liver, and from < 3 ng/g to 10.8 ng/g in muscle samples. After the 77th day of therapy QCA was found in samples of liver (9.7 ng/g). Recoveries obtained for both liver and muscle were 70% at 5 ng/g, 77% and 75% respectively at 10 ng/g, and 90% for both liver and muscle at 30 ng/g. This experiment was performed within the frame of the National Monitoring Programme of Residues in Animal Tissues in the Republic of Croatia.

Determination of Carbadox and Its Metabolite, Quinoxaline-2-carboxylic Acid, in Swine Tissues by HPLC

A simple and rapid method for the simultaneous determination of carbadox (CDX) and quinoxaline-2-carboxylic acid (QCA), the most persistent metabolite, in swine tissues has been developed by HPLC with photodiode array detection. The drugs were extracted with 0.3% metaphosphoric acid-MeOH (7:3, v/v), and the extracts were cleaned up with a Bond Elut C18 cartridge. The HPLC separation was performed on a Wakosil-II 5C18 RS column (150×4.6mm i. d.) with a gradient system of 0.01M phosphate buffer (pH 2.5)-MeCN as the mobile phase, at the flow-rate of 0.6ml/min. The calibration graphs were rectilinear from 0.5 to 50ng for CDX and QCA. The recoveries of CDX and QCA from swine muscle and liver fortified at the level of 0.1μg/g were 85.0-94.3%, with high precision. The limits of detection were 0.005-0.01μg/g for CDX and QCA. Further, a preliminary investigation was carried out in order to determine the toxic metabolite desoxy-carbadox.

Determination of quinoxaline carboxylic acid (metabolite of carbadox) in animal tissue by HPLC.

A method for the determination of residues of quinoxaline-2-carboxylic acid (QCA), the major metabolite of carbadox, in pig kidney has been developed. Tissue samples were subjected to alkaline hydrolysis, liquid-liquid extractions, ion-exchange chromatography and further extraction to concentrate the analyte and purify the extract. Determination was by reverse phase HPLC with UV detection at 320 nm. Validation exercises carried out on batches of six samples fortified with 0.050 mg/kg QCA on three separate days gave mean recoveries ranging from 72 to 79% with relative standard deviations ranging from 5.6 to 7.8%. Samples fortified with 0.010 mg/kg QCA gave a mean recovery of 100% with a relative standard deviation of 9.8%.

Confirmatory Identification of Carbadox-Related Residues in Swine Liver by Gas-Liquid Chromatography/Mass Spectrometry with Selected Ion Monitoring

Abstract A confirmatory method has been developed to identify quinoxaline-2-carboxylic acid, the carbadox tissue residue, in swine liver, a target tissue, at the regulatory level of 30 μg/kg. Quinoxaline-2-carboxylic acid (QCA) is isolated from liver hydrolysates by solvent extraction and ion-exclusion chromatography, and a methyl ester derivative (CP-25,536 or QME) is identified by gas-liquid chromatography/ mass spectrometry with selected ion monitoring. The relative intensities of 3 ions: the base peak at m/z = 130, a second significant mass at m/z = 158, and the molecular ion (M+) at m/z = 188, are monitored simultaneously with a quadrupole mass spectrometer. Validation studies consisting of the analysis of liver fortified with QCA at the regulatory level and analysis of swine specimens containing physiologically incurred carbadox residues demonstrated that peak height ratios of ions in these tissue extracts corresponded to ion intensities of standards monitored at m/z = 188,158, and 130.

Studies on the cell-free synthesis of echinomycin and an echinomycin analogue.

Abstract