Michael O'Keeffe

Analysis of Protein-bound metabolites of Furazolidone and furaltadone in pig liver by high-performance liquid chromatography and liquid chromatography–mass spectrometry

Studies undertaken using radiolabelled furazolidone have demonstrated the covalent binding of residues of the drug to cellular protein in vivo. A portion of these bound residues and those formed by furaltadone, a related nitrofuran drug, possess intact side-chains, 3-amino-2-oxazolidinone (AOZ) and 5-morpholino-methyl-3-amino-2-oxazolidinone (AMOZ), respectively. These side-chains have molecular characteristics in common with the parent compounds and may be released from liver tissue under mild acidic conditions. Derivatization with 2-nitrobenzaldehyde (NBA) serves to isolate the released side-chains and the derivatives NPAOZ and NPAMOZ are chromophoric, thereby permitting UV detection. This paper reports the introduction of an extract clean-up step to the existing procedure which eliminates or decreases interference from NBA in the HPLC-UV determination of NPAOZ. The modified procedure was also applied to the determination of AMOZ. The development of an LC-MS method for the quantitative and confirmatory determination of AOZ and AMOZ extracted and derivatized according to the same procedure as that for HPLC-UV is described. The methods were validated for AOZ and AMOZ in fortified (intra- and inter-assay studies) and incurred (inter-assay studies) pig liver samples. The limit of determination for fortified control liver samples was 5 ng AOZ g-1 and 10 ng AMOZ g-1 by HPLC-UV and 10 ng AOZ or AMOZ g-1 by LC-MS. In addition, a study to determine the ratio of released AOZ to the total bound residues present in incurred liver samples from pigs treated with furazolidone is described.

Multi-residue analysis for beta-agonists in urine and liver samples using mixed phase columns with determination by radioimmunoassay.

A method is described for the extraction of four beta-agonists, clenbuterol, salbutamol, mabuterol and terbutaline from bovine urine and liver samples using radioimmunoassay (RIA) as the method of determination. Following enzymic digestion of the liver samples using protease enzyme, the digest is centrifuged and the harvested supernatant is saturated with sodium chloride and adjusted to pH 11.0. An ethyl acetate-propan-2-ol mixture is used to extract the beta-agonists from the liver digest. The samples of urine and liver extracts are adjusted to pH 6.0 and applied to mixed phase (XtrackT) columns. The column is washed with water and methanol and the beta-agonists are eluted with methanol containing 2% ammonia. After evaporation of the eluting solvent and reconstitution in ethanol the beta-agonist residues are determined by RIA, with standard graphs prepared in residue-free sample extract. The procedure has been validated for clenbuterol, salbutamol, mabuterol and terbutaline. The mean recovery of the beta-agonists from urine and liver is > 75% and > 85%, respectively. The detection limit is 0.13 ng ml-1 and 0.46 ng g-1 of clenbuterol in urine and liver, respectively. The high recoveries attained for both types of beta-agonists are a result of an efficient liquid-liquid extraction step coupled with a selective mixed solid-phase extraction procedure.

Evaluation of the Premi® Test and comparison with the One-Plate Test for the detection of antimicrobials in kidney

The Premi® Test, a test kit designed for the rapid screening of antimicrobial residues in meat, fish and eggs, was evaluated and compared with the (modified) One-Plate Test, an agar diffusion assay. The performance characteristics described for qualitative, screening methods in Commission Decision 2002/657/EC were used for the evaluation. The Premi® Test was found to detect a range of antimicrobials to MRL levels in kidney fluid but to have poorer sensitivity for some antimicrobials such as tetracyclines, sulphonamides, flumequine and streptomycin. The test was found not to be sensitive for the banned antimicrobial chloramphenicol. The One-Plate Test was found to detect most tetracyclines and flumequine to MRL levels but to be less sensitive than the Premi® Test for most of the other classes of antimicrobials. Neither test alone provides a comprehensive screening test for antimicrobial residues in kidney at MRL levels. However, the Premi® Test is fast, easy to use and rugged and, in combination with other antimicrobial tests, may be used to provide a comprehensive screening system for antimicrobials in tissues.

Differential-pulse voltammetric determination of clenbuterol in bovine urine using a Nafion-modified carbon paste electrode

A method has been developed for the determination of clenbuterol in bovine urine using differential-pulse voltammetry (DPV), based on the electrochemical behaviour of clenbuterol at a Nafion-modified carbon paste electrode (CPE). Clenbuterol is irreversibly oxidized at high positive potentials, its irreversibility being due to a chemical follow-up reaction which results in a product showing quasi-reversible electrochemical behaviour at much lower potentials. It is the oxidation peak of this product, arising in acidic media at 0.42 V, which was analysed using DPV, again following the accumulation of clenbuterol at the Nafion-modified CPE. Electrode renewal was achieved by holding the potential at -0.6 V for 120 s in 0.1 mol l-1 NaOH. The determination of clenbuterol in the presence of interfering compounds present in bovine urine samples was then carried out after a two-step clean-up of the urine involving liquid-liquid extraction followed by a mixed-mode solid-phase extraction procedure. This allowed clenbuterol to be detected down to a level of 1.02 x 10(-9) mol l-1 in bovine urine extracts.

Matrix solid-phase dispersion as a multiresidue extraction technique for β-agonists in bovine liver tissue

A procedure for the extraction of salbutamol including conjugated forms of the drug, from liver samples, is described. It combines matrix solid-phase dispersion with radioimmunoassay for the measurement of salbutamol residues at the sub-ppb level. Inter- and intra-assay validation, carried out on fortified liver samples, show good recoveries over the range 1-5 ppb of salbutamol. An enzyme hydrolysis procedure was optimized for the deconjugation of incurred residue. The developed procedure is shown to be suitable for the extraction and determination of other beta-agonists such as clenbuterol, mabuterol, terbutaline, and cimaterol at residue levels of less than 1 ng g-1.

Reversed-phase high-performance liquid chromatographic determination of dexamethasone in bovine tissues

A sensitive and selective high-performance liquid chromatographic procedure is described for the determination of the synthetic corticosteroid dexamethasone (DXM), in bovine muscle, kidney, liver and fat tissues, using methylprednisolone as the internal standard. Following extraction with ethyl acetate (muscle, kidney and liver) or diethyl ether (fat) and clean-up of the tissue extract, the drug residue was isolated using a C18 solid-phase extraction column. Separation of DXM was achieved by reversed-phase high-performance liquid chromatography with ultraviolet detection at 254 nm. By using this procedure, DXM levels as low as 0.01 mg kg-1 can be detected in muscle, kidney, liver and fat.

Application of matrix solid phase dispersion for the determination of clenbuterol in liver samples

A rapid method for determination of clenbuterol in liver samples has been developed. It involves combining matrix solid phase dispersion with radioimmunoassay as an efficient technique for residue determination at the sub ng g−1 level. The technique was optimised for extract clean-up and recovery of residue using radiolabelled [3 H]-clenbuterol. Inter- and intra-assay validation, carried out on fortified liver samples, showed high recoveries over the range 1–5 ng clenbuterol g−1. The determination of incurred residues in liver samples has been investigated. The developed procedure allows for determination of residues of clenbuterol in tissues at levels of less than 0.5 ng g−1.

Survey of the anticoccidial feed additive nicarbazin (as dinitrocarbanilide residues) in poultry and eggs.

A survey was carried out on the occurrence of dinitrocarbanilide (DNC), the marker residue for nicarbazin, in poultry produced in Ireland during 2002–2004. Liver (n = 736) and breast muscle samples (n = 342) were tested. DNC residues were found in 40 and 26% of liver and breast muscle samples at levels greater than 12.5 and 5 µg kg−1, respectively. DNC residues were found at >200 µg kg−1 in 12 and 0% of liver and muscle samples, respectively. Samples of breast muscle (n = 217) imported from 11 countries were also tested for DNC residues. A lower incidence of DNC residues (6%) was found in imported breast muscle. Egg samples (n = 546) were tested and DNC residues were found in nine samples, with levels ranging between 14 and 122 µg kg−1. Analysis of poultry, carried out as part of official food inspection in the period 2004–2006, indicated a reduction in the number of broiler liver samples containing DNC at >200 µg kg−1, to approximately 7%. Low levels of DNC residues continue to be found in <2% of egg samples.

Investigation of the causes for the occurrence of residues of the anticoccidial feed additive nicarbazin in commercial poultry

Investigations were undertaken to identify causes for the occurrence of high levels of the zootechnical feed additive nicarbazin in broiler liver at slaughter. The first investigation on 32 commercial broiler flocks involved sampling and analysis for nicarbazin (as dinitrocarbanilide, DNC) in liver from birds during a 3–10-day period after withdrawal of nicarbazin from their feed and before commercial slaughter. DNC residues in liver samples of broilers scheduled as being withdrawn from nicarbazin for ≥6 days ranged from 20 to >1600 µg kg−1 (the specified withdrawal period for nicarbazin is 5 days and the Joint Expert Committee on Food Additives (JECFA) maximum residue limit (MRL) is 200 µg kg−1 liver). Further on-farm investigations on 12 of these flocks, selected on the basis of the feeding system in use and the levels of DNC residues determined in liver, identified issues in feed management contributing to elevated residues in broiler liver. A significant correlation (0.81, p < 0.01, n = 10) between DNC residues in liver samples and in feed samples from the feeding pans was observed. The second investigation on 12 commercial broiler flocks involved sampling and analysis for DNC in liver samples and feed samples from feeding pans and from the feed mill at the three thinnings of birds for commercial slaughter. In the case of one flock, a clear relationship between nicarbazin in feed from the feed mill (10.5 mg kg−1 DNC), in feed from the feeding pans (6.6 mg kg−1 DNC) and in liver (583 µg kg−1 DNC) at first thinning (9 days scheduled withdrawal from nicarbazin) was observed. Such a clear relationship was not observed in other cases, particularly at second and third thinnings, pointing to re-exposure of birds to nicarbazin late in the flock production cycle, probably from the litter. Guidelines outlining best farm practice to eliminate nicarbazin residues in poultry have been published in booklet and poster format for broiler producers and deal with feed system cleaning, feed bin management, feed deliveries, feed usage and records.

Studies on the persistence of estradiol benzoate and nortestosterone decanoate in hair of cattle following treatment with growth promoters, determined by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Measurement of steroid esters in bovine hair samples, using sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS), provides a powerful tool for identifying animals treated illicitly with growth promoters. The successful application of such testing requires appropriate sampling of hair from treated animals. This paper describes the results of hair analysis by LC-MS/MS for two animal studies in which animals were treated with estradiol-3-benzoate and nortestosterone decanoate. The results from the first animal study indicate that animals treated with these anabolic steroids may not always be identified from analysis of hair samples; positive test results occur sporadically and only for some of the treated animals. The results from the second animal study identify conditions attaching to positive hair samples, such as, that concentrations of steroid esters in hair are related to distance of sampling from point of injection and to time post-treatment, that concentrations of steroid esters in hair are related to dose given to the animal but that this relationship may vary over time post-treatment, and that steroid esters may be measured in regrowth hair taken some weeks after treatment. Steroid esters are determined along the length of the hair, confirming that accumulation of steroid esters into hair occurs from various sources, including blood, sweat and sebum. The reported research provides some useful insights into the mechanisms governing the persistence of steroid esters in bovine hair following illicit treatment with growth promoters.