Metabolites In Animal Tissues Research Articles

Development of a Multimatrix UHPLC-MS/MS Method for the Determination of Paracetamol and Its Metabolites in Animal Tissues.

Paracetamol/acetaminophen (APAP) is one of the most popular pharmacologically active substances used as an analgesic and antipyretic agent. The metabolism of this drug occurs in the liver and leads to the formation of two main metabolites—glucuronic acid and sulfate derivate. Despite the wide use of paracetamol in veterinary medicine, a handful of analytical methods were published for the determination of paracetamol residues in animal tissues. In this paper, a multimatrix method has been developed for the determination of paracetamol and two metabolites—paracetamol sulfate (PS) and p-Acetamidophenyl β-D-glucuronide (PG). A validation procedure was conducted to verify method reliability and fit purpose as a tool for analyzing acetaminophen and metabolites in muscle, liver, lung, and kidney samples from different species of animals. Established validation parameters were in agreement with acceptable criteria laid by the European legislation. The initial significant matrix effect was successfully reduced by implementing an internal standard—4-Acetamidophenyl β-D-glucuronide-d3 (PG-d3, IS). The usefulness of the developed method was verified by analyzing samples from an experiment in which paracetamol was administrated to geese.

Deficiency of a pyrroline-5-carboxylate reductase produces the yellowish green cocoon ‘Ryokuken’ of the silkworm, Bombyx mori

The silkworm cocoon colour has attracted researchers involved in genetics, physiology and ecology for a long time. 'Ryokuken' cocoons are yellowish green in colour due to unusual flavonoids, prolinylflavonols, while 'Sasamayu' cocoons are light green and contain only simple flavonol glucosides. We found a novel gene associated with the cocoon colour change resulting from a change in flavonoid composition and named it Lg (light green cocoon). In the middle silk glands of the + Lg /+ Lg larvae, 1-pyrroline-5-carboxylic acid (P5C) was found to accumulate due to a decrease in the activity of pyrroline-5-carboxylate reductase (P5CR), an enzyme reducing P5C to proline. Sequence analysis of BmP5CR1, the candidate gene for Lg, revealed a 1.9 kb insertion and a 4 bp deletion within the 1st intron, a 97 bp deletion within the 4th intron, and a > 300 bp insertion within the 3'-UTR, in addition to two amino acid changes on exons 3 and 4 in + Lg /+ Lg compared to Lg/Lg. Decreased expression of BmP5CR1 was observed in all of the investigated tissues, including the middle silk glands in + Lg /+ Lg , which was probably caused by structural changes in the intronic regions of BmP5CR1. Furthermore, a BmP5CR1 knockout strain exhibited a yellowish green cocoon with the formation of prolinylflavonols. These results indicate that the yellowish green cocoon is produced by a BmP5CR1 deficiency. To our knowledge, this is the first report showing that the defect of an enzyme associated with intermediate metabolism promotes the conjugation of phytochemicals derived from foods with endogenously accumulating metabolites in animal tissues.

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

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

Multiresidue analysis of 22 sulfonamides and their metabolites in animal tissues using quick, easy, cheap, effective, rugged, and safe extraction and high resolution mass spectrometry (hybrid linear ion trap-Orbitrap)

A new high performance liquid chromatography–high resolution mass spectrometry (HPLC–HRMS) method was developed for a simultaneous multi-residue analysis of 22 sulfonamides (SAs) and their metabolites in edible animal (pig, beef, sheep and chicken) tissues. Sample preparation was optimized on the basis of the “QuEChERS” protocol. The analytes were identified using their LC retention times and accurate mass; the identification was further confirmed by multi-stage high mass accuracy (<5ppm) mass spectrometry. The performance of the method was evaluated according to the EU guidelines for the validation of screening methods for the analysis of veterinary drugs residues. Acceptable values were obtained for: linearity (R2<0.99), limit of detection (LOD, 3–26μg/kg), limit of quantification (LOQ, 11–88μg/kg), accuracy (recovery 88–112%), intra- and inter-day precision 1–14 and 1–17%, respectively, decision limit (CCα) and detection capability (CCβ) around the maximum residue limits (MRL) of SAs (100μg/kg). The method was validated by analysis of a reference material FAPAS-02188 “Pig kidney” with ǀ Z-scoreǀ<0.63. The method was applied to various matrices (kidney, liver, muscle) originated from pig, beef, sheep, and chicken) allowing the simultaneous quantification of target sulfonamides at concentration levels above the MRL/2 and the identification of untargeted compounds such as N4-acetyl metabolites using multi-stage high mass accuracy mass spectrometry.

Characterization and estimation of sulfaquinoxaline metabolites in animal tissues using liquid chromatography coupled to tandem mass spectrometry

Sulfaquinoxaline (SQX) is a sulfonamide that is widely used in veterinary medicine, with a maximum residue limit (MRL) established for several food matrices. In Brazil, the MRL for liver and muscle is 100 μg kg−1 for equine, bovine, poultry and swine. This value includes not only free drug but also the sum of all metabolites. Several reports showed limitations for SQX residue analysis, especially when mass spectrometry methods were used. These limitations include poor recoveries and unacceptable accuracy responses. In this work a metabolite of SQX, present in liver and kidney samples, was identified. The structure proposed was a hydroxylated form of SQX, called SQX-OH, with an m/z of 317. SQX-OH is also produced in vitro in equine, swine and bovine liver samples. The influence of time, temperature, solvent and dehydration was evaluated in the formation of SQX-OH. Different degrees of hydroxylation were observed in matrices. The N4-acetyl derivates for both SQX and SQX-OH were also detected. In equines, the metabolism of SQX is complete. The mass spectrometry analysis of SQX-OH was determined in vitro using equine liver microsomal fraction. The characterization of this compound was performed using liquid chromatography coupled to mass spectrometry in tandem mode. The fragmentation profile of SQX-OH was seen to be similar to that of the sulfonamides group, producing two high abundant daughter ions: 317 > 156 and 317 > 108, common to most sulfonamides. The main conclusion of this work is that the residue analysis of SQX needs to consider the presence of the SQX-OH in order to give more realistic results, especially when using MRM transitions.

Development of Liquid Chromatographic Methods for Determination of Quinocetone and Its Main Metabolites in Edible Tissues of Swine and Chicken

Quinocetone (QCT), a new antimicrobial growth promotant of quinoxalines, can effectively improve the growth and feed efficiency of food animals with more safety than is provided by olaquindox and carbadox. To clarify its metabolism and residue levels in animals, a liquid chromatographic method with UV-Vis detection was developed for the determination of QCT and its main metabolites, desoxyquinocetone (DQCT) and 3-methylquinoxaline-2-carboxylic acid (MQCA), in muscle, liver, kidney, and fat of swine and chicken. For sample pretreatment, QCT and DQCT were extracted with ethyl acetate and purified with iso-octane; after alkaline hydrolysis of the tissue, MQCA was extracted with ethyl acetate and citric acid buffer (pH 6.0), and the extract was purified over a cation-exchange column (AG MP-50 resin). Detection was at 312 and 320 nm for QCT and DQCT, respectively, and at 320 nm for MQCA. The observed limit of detection for the 3 compounds was 0.025 microg/g in various tissues. The methods were linear over the concentrations range of 0.01-0.64 microg/mL with mean recoveries of approximately 71-86% and relative standard deviations of about 4-12% at the levels of 0.05, 0.10, and 0.20 microg/g. The method is highly selective and can be applied to the determination of QCT and its main metabolites in animal tissues, which would accelerate the pharmacokinetic and residue study of QCT in food animals.

Quantitative determination of salicylic acid and metabolites in animal tissues by liquid chromatography–tandem mass spectrometry

In order to study the excretion pattern of salicylic acid and its metabolites in animals, a liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed. The method was used to study the biotransformation of salicylic acid in poultry (chicken and pigeon) after therapeutic use, i.e. in matrices such as plasma, excreta (=combined urine and faeces), kidney and liver. Thereafter, the method was also adopted for the analysis of residues of salicylic acid in porcine tissues, such as muscle, kidney, liver and skin + fat. For the biotransformation study, salicylic acid, the oxidation and amino acid conjugation metabolites such as gentisic acid, salicyluric acid, and a double conjugated ornithine metabolite, together with the internal standard phenoxyacetic acid were extracted from 1.0 g of tissue or 100 μl of plasma into ethyl acetate, after acidifying using 1 M HCl. After centrifugation and for kidney and liver tissue, the analytes were back-extracted and subjected to a solid-phase clean-up on SAX columns. For the analysis of excreta, the sample preparation included only a dilution step, performed before LC–MS/MS analysis. For the residue study, salicylic acid and the internal standard phenoxyacetic acid were extracted from 1.0 g of porcine muscle, liver, kidney and skin + fat with 6 ml of diethyl ether, after acidifying using 3 ml of 1 M HCl. After extraction and centrifugation, the ether phase was evaporated under N 2 at 40 °C. The residue was redissolved with 500 μl of 0.1% acetic acid in water. For skin + fat and when needed for the other tissues, a supplemental extraction of the redissolved residue with 500 μl of hexane was performed. The analysis of the extracts was done on a Nucleosil 100-5 C18 column, using a gradient elution with 0.1% acetic acid in water and methanol. A Quattro Ultima ® triple quadrupole instrument was used, equipped with an ESI z-spray source, which was operated in the negative ion MS/MS mode. The methods were validated for the linearity (100–1000 ng ml −1 and 10–50 μg ml −1 for plasma; 5–250 μg g −1 for excreta; 100–1000 ng g −1 and 5–25 μg g −1 for the biotransformation study in poultry kidney and liver; 25–1000 ng g −1 for the residue study in porcine tissues); trueness and precision; specificity; limit of detection and limit of quantification. The limit of quantification for the residue analysis of salicylic acid in porcine tissues was set at 50 ng g −1.

Quantitative analysis of amoxycillin and its major metabolites in animal tissues by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

A sensitive and specific method for the quantitative determination of amoxycillin and its major metabolites (amoxycilloic acid, amoxycillinpiperazine-2',5'-dione) in animal tissue samples using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is presented. A liquid extraction using an aqueous 0.01 M potassium dihydrogenphosphate solution as the extraction solvent was performed for a preliminary sample cleanup. The extracts were further purified by a solid-phase extraction using an octadecyl (C18) column. Ampicillin was used as the internal standard. Chromatographic separation of the analytes of interest was achieved on a reversed-phase Hypersil column (100 x 3 mm i.d., dp, 5 microm), using a mixture of 9.6 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. Gradient elution was performed. To obtain as high sensitivity and selectivity as possible, the mass spectrometer was operated in the multiple reaction monitoring mode. The method was validated for the analysis of amoxycillin and its investigated metabolites in various porcine tissues, kidney, liver, muscle, and fat, according to the requirements defined by the European Community. Calibration graphs were prepared for all tissues, and good linearity was achieved over the concentration range tested (25-500 ng/g, r > or = 0.9974, and goodness of fit < or = 9.6). A limit of quantification of 25 ng/g was obtained for amoxycillin and its metabolites in all tissues, which corresponds to half the maximum residue limit for amoxycillin. Limits of detection ranged from 2.3 to 12.0 ng/g for amoxycillin and from 1.1 to 15.1 ng/g and 0.2 to 2.4 ng/g for amoxycilloic acid and amoxycillinpiperazine-2',5'-dione, respectively. The results for the within-day precision and the trueness fell within the ranges specified. The method has been successfully used for the quantitative determination of amoxycillin and its major metabolites in tissue samples from pigs medicated via the drinking water, proving the usefulness of the developed method for application in the field of residue analysis.

Gas chromatographic determination of organophosphorus insecticides and their dialkyl phosphate metabolites in liver and kidney samples

A method is described for the quantitative determination of organophosphorus insecticides and six dialkyl phosphate metabolites in animal tissues. The procedure relies on solvent extraction, automated gel permeation chromatography cleanup, and gas chromatographic analysis with flame photometric detection. Tetrabutylammonium hydroxide is utilized as the derivatization reagent for dialkyl phosphate metabolite analysis. Method detection limits ranged from 0.02 to 0.05 ppm for the compounds studied. The method was applied to the analysis of liver and kidney samples of pigeons exposed to diazinon and parathion. Residue levels were confirmed by mass spectrometry, using selected ion monitoring.

Release and characterization of bound benomyl and carbendazim metabolites in animal tissues via Raney nickel desulfurization and acid dehydration

Release and characterization of bound benomyl and carbendazim metabolites in animal tissues via Raney nickel desulfurization and acid dehydration

Biological Evaluation of Aflatoxins and Metabolites in Animal Tissues

Biological Evaluation of Aflatoxins and Metabolites in Animal Tissues

Analysis of veterinary residues in foods

Increasing world population, and particularly the urbanisation of that population, has meant that efforts are constantly being made to increase food production. This enhanced productivity, especially using intensive rearing practices as in poultry, eggs and veal production, has been accompanied by, and in many cases made possible by, the use of chemicals (Fig. 1). These may take the form of drugs to prevent or cure disease, feed additives or chemical implantation for growth promotion, tranquillisers to reduce stress before slaughter and oestrous-synchronising agents to control breeding or milk production. Consequently, worries have arisen about chemical residues in food and their possible toxic effects. The problem was addressed as early as 1969 in the U.K. with the publication of the Swann report’ in which the use of antibiotics in food production was investigated following fears of bacterial resistance building up in the animal’s gut flora possibly passing antibiotic resistance onto bacteria either indigenous to man or, perhaps more importantly, pathogenic to man. Also, antibiotic residues present in animal products could result in hypersensitivity in some people, and the development of a reservoir of antibiotic resistance in man’s natural bacterial flora. Many countries now have laws governing the use of veterinary drugs and drug residues in foods. Regulations vary from country to country reflecting differences in disease patterns and animal husbandry practices. A recent report2 of a joint FAO/WHO expert consultation on veterinary drug residues stresses the need for the establishment of internationally agreed maximum residue limits (MRLs) so that food products are not excluded from world markets. Commodities for which MRLs are needed must be identified and agreements on the residue data for establishing MRLs made. Further problems include the difficulty of identification of drug residues and their metabolites in animal tissues, their quantitation at levels well below the MRLs, and the need to establish and control withdrawal periods before slaughter. Drug residues can occur in all edible products of treated animals and analytical methods must be able to cope with different levels of fat and other interfering compounds which may be present. Recent pharmacokinetic studies2 have shown that a rapid initial fall in applied drug concentration is followed by a much slower phase of depletion, emphasizing the importance of withdrawal periods and the need for extremely sensitive and selective techniques for the identification and quantitation of residues. Two types of analytical approach are required. The first, the most cost effective, consists of rapid screening techniques for presumptive evidence of the nature and levels of substances present. Screening methods should be amenable to both multiresidue analysis and use outside the laboratory. Unequivocal identification and quantitation of suspected positive samples using more expensive instrumental techniques forms the second approach.

Analysis of S-adenosylmethionine and related sulfur metabolites in animal tissues

A high-performance liquid chromatographic method was devised that separates S-adenosylmethionine and related sulfur metabolites on a Radial-PAK SCX cation-exchange column using a four-step NH 4COOH (NH 4) 2SO 4 elution gradient. This new procedure permits, in a single run of 60 min, the quantitative analysis of S-adenosylmethionine, S-adenosylhomocysteine (AdoHcy), 5′-deoxy-5′-methylthioadenosine, decarboxylated S-adenosylmethionine, decarboxylated AdoHcy, inosylhomocysteine, and other related metabolites. Furthermore, this method allows the detection in rat tissues of novel sulfur metabolites, S-inosylhomocysteine and decarboxylated AdoHcy. Perturbation of the levels of some of these metabolites could be detected in rat livers and spleens after the administration of 3-deazaadenosine, an inhibitor of AdoHcy hydrolase, but could not be detected in rat adrenal glands. It is notable that decarboxylated AdoHcy disappeared in the livers of rats treated with 3-deazaadenosine. HeLa cells incubated with [ 35S]methionine displayed the incorporation of the labeled sulfur into S-adenosylmethionine, AdoHcy, decarboxylated S-adenosylmethionine, S-inosylhomocysteine, and 5′-deoxy-5′-methylthioadenosine.

High Performance Liquid Chromatographic Quantitation of Aflatoxin Metabolites in Animal Tissues

A previously reported reverse phase high performance liquid chromatographic (HPLC) procedure was modified to permit the determination of the parent aflatoxins and various free and conjugated metabolites in animal tissues. The modified procedure was based on HPLC analysis of duplicate portions of a sample extract which had been prepared with and without treatment with trifluoroacetic acid (TFA). TFA catalyzes the conversion of aflatoxins G1, B1, M1, and Q1 to the fluorescent derivatives G2a, B2a, M2a, and Q2a. Aflatoxicol, which exhibited a sharp fluorescent peak in its native state, eluted as a tailing peak with weaker fluorescence following TFA treatment. Acid hydrolysis of the aqueous phase of tissue samples permitted the analysis of water-soluble conjugated forms of the various aflatoxins. Analysis of fluorescence spectra of manually collected HPLC fractions qualitatively supported the accuracy of the method. Representative data on the distribution of aflatoxins in turkey liver following ingestion of an experimentally contaminated ration are presented.

Gas chromatographic residue method for iodofenphos and two of its metabolites in animal tissues, fat and milk

AbstractA gas chromatographic residue method for use on animal tissues, fat, and milk is presented for the organophosphorus insecticide iodofenphos. The procedure includes the determination of two potential metabolites, iodofenoxon, and 2,5‐dichloro‐4‐iodophenol. The two phosphorus‐containing compounds are analysed by means of a flame photometric detector after cleanup including silica gel column and thin‐layer chromatography. The substituted phenol is determined separately as the acetylated derivative using an electron capture detector. Some information on the problem of specificity with regard to possible interferences by closely related insecticides or their corresponding phenols is also given.

A New Oxidation Catalyst

COENZYMES I and II occupy pivotal positions in biological oxidations. The great majority of metabolites in animal tissues are oxidized through the intermediation of these coenzymes. But whereas the mechanism of the reduction of the coenzymes by dehydrogenase systems is well understood, the physiological mechanism of the oxidation of reduced coenzyme I or II has been obscure.