Metabolite profiling using liquid chromatography/quadrupole time-of-flight mass spectrometry for the identification of a suitable marker and target matrix of griseofulvin use in bovines.

RATIONALE: Griseofulvin is an antifungal agent with potential for misuse in food-producing animals. Little is known about its metabolism in ruminants and hence what are suitable marker residues and target matrices for monitoring purposes. METHODS: Tissues harvested from cattle treated with the antifungal agent griseofulvin were screened using liquid chromatography coupled to positive and negative electrospray ionization (ESI) quadrupole time-of-flight mass spectrometry (qToFMS) operated in ToF mode. RESULTS: Twenty-five possible metabolites were detected across all tissue types, but two isomeric compounds with accurate masses corresponding to loss of a methyl group from parent griseofulvin were considered to be the best candidate markers. Data from fragmentation experiments enabled a tentative assignment of the structures of the two compounds as 4-demethylgriseofulvin and 6-demethylgriseofulvin. These assignments were confirmed by matching the product ion spectra of incurred residues to those of custom synthesized reference standards. CONCLUSIONS: 4-Demethyl- and 6-demethylgriseofulvin have been identified as potential marker compounds of griseofulvin use in cattle. Liver was identified as the target matrix. Hair was shown to have potential for non-invasive testing.

Analysis of incurred crystal violet in Atlantic salmon (Salmo salar L.): comparison between the analysis of crystal violet as an individual parent and leucocrystal violet and as total crystal violet after oxidation with 2,3-dichloro-5,6-dicyanobenzoquinone.

Due to on-going concern about the occurrence of triphenylmethane dye residues in fish destined for human consumption, a depletion study of crystal violet in salmon was carried out. Atlantic salmon less than 12 months old were exposed to crystal violet in fresh water at 15°C and subsequently sampled at 1, 7, 14, 28, 63 and 91 days after exposure. The salmon were then analysed by two analytical methods. In the first method, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) was used to oxidise leucocrystal violet to its parent form. Total parent crystal violet was then analysed by LC-MS/MS. In the second method, crystal violet and leucocrystal violet were analysed individually by LC-MS/MS without oxidation. Both methods gave comparable results for total crystal violet concentrations, with a correlation of r(2)=0.69. Statistical treatment for 88 incurred salmon samples showed no significant difference between the two sets of results with t=1.68 and t(crit)=1.99. Up to 98% of crystal violet was metabolised to its leuco form in the salmon after 1 day of exposure and could be detected at significant concentrations (approximately 20 µg kg(-1)) 91 days after exposure. The depletion data also suggest that crystal violet has a half-life of approximately 15-16 days in salmon.

Multi-residue determination of phenolic and salicylanilide anthelmintics and related compounds in bovine kidney by liquid chromatography-tandem mass spectrometry.

This paper describes an analytical method for four phenolic and salicylanilide anthelmintics authorised for use within the EU (nitroxinil, oxyclozanide, rafoxanide and closantel) in bovine kidney, and the extension of this procedure to include a number of related compounds; ioxynil, niclosamide, salicylanide and 3-trifluoromethyl-4-nitrophenol (TFM). The method comprises a solvent extraction with 1% acetic acid in acetone and clean-up using a mixed-mode anion-exchange solid phase extraction column. Determination is by reversed phase LC-MS/MS. The method was validated to the latest EU requirements (Commission Decision 2002/657/EC) using both spiked and incurred tissues and was subject to second laboratory evaluation.

The effect of cooking on veterinary drug residues in food: nicarbazin (dinitrocarbanilide component).

The change of concentration of residues of the marker compound for the anti-coccidial drug nicarbazin, N,N'-bis(4-nitrophenyl)urea (dinitrocarbanilide, DNC), was investigated in model oil and aqueous solutions and in chicken muscle and egg. In model aqueous solutions, DNC decreased rapidly in concentration upon heating followed by a much more gradual decomposition. The curves produced when this information was plotted were not typical of exponential decay. In model cooking oil solutions, DNC generally showed a slower decrease in concentration over time when compared with aqueous solutions. DNC residues in egg were stable to microwave cooking and residues in chicken muscle were stable to stewing and microwaving. Other cooking procedures led to a decrease in amount of DNC by 22% to 48% of the total amount of analyte present. Only a small amount (<2%) of residue leached with juices which exuded as the food was cooked.

Meeting maximum residue limits: an improved screening technique for the rapid detection of antimicrobial residues in animal food products.

A rapid, high-throughput antimicrobial screening assay was developed using either a physical fluid extraction or a solvent extraction technique coupled to the commercially available PremiTest. The solvent extraction approach was fully validated for a wide range of tissues and the fluid extraction approach partially validated for porcine muscle. Both procedures can detect a wide range of antimicrobial compounds at or below maximum residue limit concentrations. The use of a solvent extraction provides an enhanced test capable of detecting a wider range of drugs than the fluid extraction approach at or below half maximum residue limit levels in a variety of matrices. Biochemical methods for the class-specific identification of beta-lactams and sulphonamides following initial screening were developed and validated. The approach is a significant improvement on existing methodologies as a tool for residues monitoring in surveillance programmes.

Improved clean-up for the determination of lasalocid in 'difficult' food matrices.

Methodology has been developed for the determination of lasalocid in analytically 'difficult' matrices such as processed and spiced foods. The procedure was based on an existing silica-based solid-phase extraction (SPE) clean-up to which was added a novel NH2 SPE step before HPLC with fluorescence detection. Use of the additional step enabled the determination of lasalocid in matrices such as baby food, meat pies ('pasties'), etc. Analysis of these matrices was not possible using the standard clean-up on its own. Chromatography showed a massive reduction in the amount of co-extractives and interferences. Validation data were obtained down to the 10-40 mg kg(-1) level for a range of products. Recoveries ranged from 74% at 10 microg kg(-1) for pork sausages to 96% at 40 microg kg(-1) for meat pies.

Screening of sulphonamides in egg using gas chromatography-mass-selective detection and liquid chromatography-mass spectrometry.

A rapid extraction and clean-up procedure for sulphonamide antibiotics in eggs suitable for both GC-MSD and LC-MS end determinations has been developed. The drugs were extracted using acetonitrile, acidified using acetic acid and cleaned-up using cation- and anion-exchange. For determination by GC-MSD, extracts were derivatised with diazomethane followed by pentafluoropropionic acid anhydride. For LC-MS extracts were taken up in water and used directly. The methodology developed was validated at the 100 and 25 microg kg(-1) levels, equivalent to the MRL and one quarter of the MRL. Results for the GC-MS procedure were quantitated against deuterated sulphadiazine with relative recoveries ranging from 54% for sulphachloropyridazine to 135.5% for sulphamethazine. Recoveries for the LC-MS procedure ranged from 33% for sulphaguanidine to 92% for sulphamethazine and sulphadimethoxine.

Improved method for the on-line metal chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products.

An improved on-line metal chelate affinity chromatography-high-performance liquid chromatography (MCAC-HPLC) method for the determination of tetracycline antibiotics in animal tissues and egg has been developed. Extraction was carried out with ethyl acetate. The extract was then evaporated to dryness and reconstituted in methanol prior to on-line MCAC clean-up and HPLC-UV determination. Recoveries of tetracycline, oxytetracycline, demeclocycline and chlortetracycline in the range 42% to 101% were obtained from egg, poultry, fish and venison tissues spiked at 25 micrograms kg-1. Limits of detection less than 10 microgram kg-1 were estimated for all four analytes. This method has higher throughput, higher recovery and lower limits of detection than a previously reported on-line MCAC-HPLC method which involved aqueous extraction and solid-phase extraction clean-up.

Screening and confirmation of triphenylmethane dyes and their leuco metabolites in trout muscle using HPLC-vis and ESP-LC-MS.

An extraction and clean-up protocol for the determination of Malachite Green and Crystal Violet and the corresponding leuco compounds in trout muscle has been developed. Final determination is by HPLC with visible (screening) or ESP-MS (confirmation) detection. In both cases lead(IV) oxide was used on-line to oxidise the leuco compounds back to the parent after chromatographic separation and prior to detection. The procedure was validated down to 2 micrograms kg-1. Intra- and inter-batch precision was measured at 3 levels for all compounds. Recoveries were in the range 66-116% with RSD of 1-17% for determination by HPLC with visible detection. For LC-MS determination, recoveries were in the range 61-94% with RSD of 4-15%. Limited surveillance data indicated that Malachite Green usage was more effectively monitored by including the leuco compound as well as the parent (9 positives for the leuco compound as opposed to 1 for Malachite Green out of 31 samples analysed).

Aspects of extraction, spiking and distribution in the determination of incurred residues of chloramphenicol in animal tissues.

Studies of distribution, extraction procedures and spiking protocols in the determination of incurred chloramphenicol residues in animal tissues have been carried out. An extraction procedure involving glucuronidase enzyme digestion was found to extract 10 times more incurred chloramphenicol from pig kidney than direct extraction without digestion. However, neither protease digestion nor ultrasonic probe treatment resulted in improved chloramphenicol extraction. Chloramphenicol was found to be inhomogeneously distributed within kidney from a treated pig. Highest concentrations were detected in the renal medulla. Muscle tissues from the same animal were found to contain a lower concentration of chloramphenicol residues, but no chloramphenicol residues were detectable in the liver. Chloramphenicol recovery from spiked pig liver was found to be lower than that from kidney, but was improved by the addition of piperonyl butoxide before extraction. This additive had no effect on recovery from spiked pig or cattle kidney. The implications of these results for regulatory surveillance of animal tissue for chloramphenicol residues are discussed.

Effects of extraction and spiking procedures on the determination of incurred residues of oxytetracycline in cattle kidney.

The effects of different extraction and spiking procedures on the determination of incurred oxytetracycline residues in animal tissues have been investigated. The extraction procedures investigated--direct aqueous or organic solvent extraction, enzymic digestion or sonication--all gave similar results for incurred oxytetracycline concentration in cattle kidney after correction for spike recovery. There was therefore no evidence for binding or conjugation of oxytetracycline in this tissue. Highest recovery from spiked tissue was obtained using ethyl acetate as extractant. The effects of spiking procedure (spike contact time, spike solvent and tissue state) on recovery from spiked cattle kidney were also small, indicating that added oxytetracycline spike does not interact with the tissue.

On-line metal chelate affinity chromatography clean-up for the high-performance liquid chromatographic determination of tetracycline antibiotics in animal tissues.

An on-line high-performance liquid chromatographic (HPLC) method for the determination of tetracycline, oxytetracycline, chlortetracycline and demeclocycline using metal chelate affinity chromatography-reversed-phase HPLC has been developed. The drugs were extracted with succinate buffer and the extract diluted with EDTA-pentanesulphonate buffer. Diluted extract was then absorbed onto a C8 or XAD-2 solid-phase extraction (SPE) cartridge and eluted with methanol. The eluate was then injected onto a TSKgel chelate column which had been preloaded with copper(II). The tetracyclines were eluted from this column onto the analytical column (Polymer Labs. PLRP-S) with an EDTA-containing buffer. Elution of the analytical column was via a methanol-acetonitrile gradient and detection was by UV at 350 nm. Average recoveries at the 10, 20, 50 and 300 micrograms kg-1 levels were 50-80%. The limit of detection (LOD) was 10 micrograms kg-1 for oxytetracycline and tetracycline and 20 micrograms kg-1 for chlortetracycline and demeclocycline. The method was validated for sheep liver and cattle kidney.

Development of multi-residue methodology for the HPLC determination of veterinary drugs in animal tissues.

The application of solid-phase extraction and HPLC with UV-diode array detection to the multi-residue determination of veterinary drugs is described. A two-stage SPE clean-up was employed, using C18 and silica cartridges. HPLC analysis was carried out on a base-deactivated C8 column using gradient elution systems at two pH values. The procedure establishes the basis of a method for routine screening of pig kidney samples for some sulphonamides, benzimidazoles, nitroimidazoles and nitrofurans at concentrations at or below the UK Maximum Residue Limits (MRLs). Limits of detection of 2-18 micrograms/kg could be achieved for these analytes at recoveries of 40-70%. UV spectra measured on-line were used for confirmation of peak identities at these concentrations. The possibility of extension of this procedure to a wider range of analyses is discussed.

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%.

High-performance liquid chromatographic method for the determination of the anthelmintic nitroxynil in cattle muscle tissue with on-line anion-exchange clean-up.

A high-performance liquid chromatographic method for the determination of the anthelmintic nitroxynil has been developed. The drug was extracted from cattle muscle tissue with 1% triethylamine in acetonitrile. The extract was evaporated to dryness and taken up in 0.1 M ammonium acetate-acetonitrile (50:50, v/v). The extract was then injected onto a polymeric anion-exchange precolumn. After clean-up with 0.1 M ammonium acetate-acetonitrile (50:50, v/v) for 5 min, the precolumn was eluted with 1% aqueous trifluoroacetic acid-acetonitrile (50:50, v/v) onto a PLRP-S polymer column and chromatographed with a mobile phase of 0.01 M phosphate pH 7-acetonitrile (80:20, v/v). Detection was by ultraviolet at 273 nm. Average recoveries at four levels from 0.005 to 1.000 mg kg-1 were > 88%. The limit of determination was 0.005 mg kg-1.

High-performance liquid chromatographic method for the determination of the dinitrocarbanilide component of nicarbazin in eggs with on-line clean-up.

A high-performance liquid chromatographic method for the determination of the dinitrocarbanilide component of the anti-coccidial drug nicarbazin has been developed. The drug was extracted from egg with acetonitrile. The extract was evaporated to dryness and taken up in water-acetonitrile (80:20, v/v). The extract was then injected onto a reversed-phase precolumn. After clean-up with 20% aqueous acetonitrile for 5 min, the precolumn was eluted onto a Chromspher C18 cartridge column with 0.01 M potassium dihydrogen-phosphate pH 4.0-acetonitrile (50:50, v/v). Detection was by ultraviolet at 343 nm. Average recoveries at five levels from 0.005 to 0.500 mg kg-1 were > 80%. The limit of determination was 0.005 mg kg-1.

Improved high-performance liquid chromatographic procedure for the determination of lasalocid in chicken tissues and egg using polymeric and porous graphitic carbon columns.

A high-performance liquid chromatographic (HPLC) method for the determination of the ionophore coccidiostat lasalocid in poultry muscle and eggs was developed. The drug was extracted from tissue with acetonitrile. The extract was partitioned between saturated salt and carbon tetrachloride and the organic layer evaporated to dryness. Clean-up was by solid-phase extraction on a silica column. HPLC analysis was carried out on either a polymeric PLRP-S or a porous graphitic carbon Hypercarb column with a basic mobile phase and fluorescence detection with excitation at 310 nm and emission at 420-430 nm. Average recoveries from poultry muscle at the 0.002, 0.010 and 0.050 mg kg-1 levels were 65.7, 72.0 and 77.9%, respectively. Average recoveries from egg at the 0.010 and 0.100 mg kg-1 levels were 76.2 and 76.4%, respectively.

Analysis of enrofloxacin and its metabolite ciprofloxacin in bovine and porcine muscle by high-performance liquid chromatography following cation exchange clean-up.

A simple and rapid method of analysis for the trace residue determination of enrofloxacin and its metabolite ciprofloxacin has been developed. Clean-up of the samples is by cation exchange solid phase extraction (SPE) and determination made by high-performance liquid chromatography using a base-deactivated column and fluorescence detection. The method has been validated for the determination of residues in bovine and porcine muscle tissue and bacon. Recoveries at the 0.010 mg kg-1 level for enrofloaxacin and ciprofloxacin respectively were 90%, 75% in bovine muscle, 75%, 54% in porcine muscle and 81%, 63% in bacon. Determination to the 0.001 mg kg-1 level in bovine muscle and to the 0.002 mg kg-1 level in porcine muscle and bacon was also carried out. The method has been used as a quantitative screening procedure.

High-performance liquid chromatographic determination of imidocarb in cattle kidney with cation-exchange clean-up.

A high-performance liquid chromatographic (HPLC) method for the determination of the antiprotozoal agent imidocarb in cattle kidney is developed. The drug is extracted from tissue with acetone in the presence of base. The extract is partitioned between saturated salt and chloroform and the organic layer evaporated to dryness. Clean-up is by cation-exchange solid-phase extraction on a carboxylic acid column. HPLC analysis is carried out on a Spherisorb S3W-PC18 column with ultraviolet detection at 260 nm. Average recoveries at the 0.05 and 0.01 mg kg-1 levels are 77.5 and 76.3%, respectively. The limit of detection is 0.001 mg kg-1.