The occurrence of nitrofuran metabolites in the tissues of chickens exposed to very low dietary concentrations of the nitrofurans

The global problem of food products contaminated by residues of the banned carcinogenic nitrofuran drugs has prompted research into how such residues accumulate in tissues. In the study described here, two aspects have been investigated where the nitrofurans accumulate in tissues from chickens exposed to either a dietary or an environmental source of contamination. Twenty groups of broilers were fed a diet containing one of the nitrofurans: furazolidone, nitrofurazone, nitrofurantoin or furaltadone at concentrations of 30, 100, 300, 1000 and 3000 µg kg^-1. At the lowest concentration of furazolidone contamination (0.01% of the therapeutic dose) tissue bound AOZ metabolite residues were detected in liver (1.1 ± 0.2 µg kg^-1) and in muscle (0.33 ± 0.03 µg kg^-1). Similar results were obtained for AMOZ (0.6 ± 0.2 µg kg^-1 in liver), the tissue bound metabolite of furaltadone. There was no appreciable accumulation of nitrofurantoin in chicken muscle. The AHD metabolite was not detected in muscle from birds fed nitrofurantoin at either 30 or 100 µg kg^-1. For nitrofurazone the concentrations of the SEM metabolite were higher in muscle than in liver for all dietary concentrations. The potential for a contaminated environment to cause nitrofuran residues in chickens was investigated. Six chickens were placed in a pen that was previously occupied by birds fed a diet containing 3000 µg kg^-1 of furazolidone. After 24 hours' exposure of the chickens to the litter in the pen, AOZ residues of 0.13 ± 0.04 and 0.10 ± 0.03 µg kg^-1 were detected in liver and muscle, respectively. The results of both experiments have implications for the poultry industry in trying to eliminate nitrofurans from their production systems, and for regulatory analysts faced with the detection of low concentrations of the drugs, both in tissues and in feedingstuffs.     

Comparison of various assays used for detection of beta-lactam antibiotics in poultry meat

In this study, microbiological tests for the detection of beta-lactam antibiotics in meat and meat products were evaluated. The traditional FPT (four plate test, containing Bacillus subtilis and Kocuria rhizophila), BsDA (Bacillus stearothermophilus disc assay) and a newly developed microbiological test, Premi®Test (containing Bacillus stearothermophilus) were included in the study. The limit of detection (LOD) of the Premi®Test was compared with the LOD of the traditional methods. The detection limits of the tests were determined by using beta-lactam antibiotic standards dissolved in meat juice, as well as meat tissue obtained from laying hens after experimental administration of amoxicillin. Positive samples, based on inhibition of growth of the organism in the test, were confirmed by high performance liquid chromatography (HPLC). Growth inhibition in the traditional tests is visible as a clear zone on the plate, whereas for Premi®Test, this is based on the absence of a colour change of the test. The LODs of antibiotics tested were as follows: Penicillin G (PENG) 5 µg kg^-1, amoxicillin (AMOX) 10 µg kg^-1, ampicillin (AMP) 25 µg kg^-1, oxacillin (OXA) 30 µg kg^-1, and cloxacillin (CLOX) 30 µg kg^-1 on the plate with Bacillus stearothermophilus. Beta-lactam antibiotics can be detected also on one plate seeded with Kocuria rhizophila, although the LODs are higher: PENG 10 µg kg^-1, AMOX 25 µg kg^-1, AMP 30 µg kg^-1, OXA 50 µg kg^-1, and CLOX 50 µg kg^-1. Premi®Test was performed according to the Standard Operating Procedure intended for detection of beta-lactam antibiotics in poultry tissues with following LODs: PENG 4 µg kg^-1, AMOX 5 µg kg^-1, AMP 5 µg kg^-1, OXA 40 µg kg^-1, CLOX 50 µg kg^-1. All tests are able to detect beta-lactam antibiotics such as penicillin G, ampicillin, amoxicillin, oxacillin and cloxacillin below the maximum residue level (MRL). However, the detection limits of the Premi®Test for PENG, AMOX and AMP were below the limits of BsDA and the plate containing Kocuria rhizophila.     

An intercomparison study of the determination of glyphosate, chlormequat and mepiquat residues in wheat

An intercomparison study of the determinations of glyphosate, chlormequat and mepiquat residues in cereals was performed. Four samples comprising one blank, two incurred and one spiked sample were sent to six participating laboratories. For glyphosate, two laboratories reported considerably lower results than the other four. One of the two laboratories with low results also reported low recoveries. The results of a sample spiked with 0.80 mg kg^-1 glyphosate and an incurred sample, ranged from 0.23-0.87 mg kg^-1 and 0.11-0.25 mg kg^-1 respectively. The strong correlation between the two samples (r² = 0.95) indicates a systematic between-laboratory variation. Several different principles were used for the analysis of glyphosate using different clean-up techniques and GC/MS, HPLC-fluorescence or LC/MS for detection. The results of the chlormequat residues showed more consistency. All but one laboratory obtained comparable results. However the correlation between the results for the sample spiked with 0.38 mg kg^-1 (range: 0.26-0.65 mg kg^-1) and the incurred samples (range: 0.19-0.45 and 0.15-0.23 mg kg^-1, respectively) again showed a strong correlation (r² = 0.99 and 0.88) indicating a systematic component. For mepiquat, results above the limit of quantification were only reported for the spiked sample. The results ranged from 0.29-0.92 mg kg^-1 (spiked concentration = 0.38 mg kg^-1). Three laboratories had results that deviated less than 25% from the fortified concentration. Two laboratories reported results 38% and 141% above the fortified concentration, respectively.     

Australian survey of acrylamide in carbohydrate-based foods

A method was developed and validated for the determination of acrylamide in carbohydrate-based foods. Solid-phase extraction employing a mixed-bed anion and cation exchange cartridge in series with a C18 extraction disk was used to clean-up water extracts of food samples before analysis by liquid chromatography coupled with tandem mass spectrometry detection. The limit of detection was calculated as approximately 25 μg kg^-1 and the limit of reporting was 50 μg kg^-1. The average method recovery for 84 samples from a range of matrices reporting was 99% with a relative standard deviation of 11.2%. A survey was conducted of 112 samples of carbohydrate-based foods composited from 547 products available in the Australian market. The analytical results were used in conjunction with Australian food consumption data derived from the 1995 National Nutrition Survey (NNS) to prepare preliminary dietary exposure estimates of Australians to acrylamide through only the food groups examined. Mean dietary exposure to acrylamide resulting from consumption of the foods tested, for Australians aged 2 years and above, was estimated as 22-29 µg day^-1 (equivalent to 0.4-0.5 µg kg^-1 bodyweight day^-1) and between 73 and 80 µg day^-1 (1.4 and 1.5 µg kg^-1 bodyweight day^-1) for 95th percentile consumers. Young children (2-6 years) consuming acrylamide-containing foods had a higher acrylamide exposure on a per kilogram bodyweight basis (mean 1.0-1.3 µg kg^-1 bodyweight day^-1). The estimated exposure of Australians to acrylamide is similar to that estimated for other countries.     

Five-year survey of ochratoxin A in processed sultanas from Turkey

The results of surveillance for ochratoxin A (OTA) in 1885 samples of sultanas taken during five crop years between 1999 and 2003 are reported. The analytical method was based on extraction with methanol + sodium bicarbonate and clean-up by immunoaffinity column chromatography followed by high-performance liquid chromatography with fluorescence detection. The limit of detection for OTA was 0.3 µg kg^-1. The results show that 9.3% of the samples contained no detectable levels of OTA, whereas 0.6% had concentrations exceeding 10 µg kg^-1; the remaining 90.3% had levels within the range 0.3-10 µg kg^-1. The overall mean OTA concentration in the total number of 1885 samples taken was 1.36 ± 2.91 µg kg^-1; the overall median was calculated as 0.90 µg kg^-1.     

Validation of an analytical method for the determination of spiramycin, virginiamycin and tylosin in feeding-stuffs by thin-layer chromatography and bio-autography

An inter-laboratory validation was carried out to determine the performance characteristics of an analytical method based on thin-layer chromatography (TLC) coupled to microbiological detection (bio-autography) for screening feed samples for the presence of spiramycin, tylosin and virginiamycin. Twenty-four samples including blank samples and samples with concentrations of the target analytes ranging between 1 and 5 mg kg^-1 (expressed in microbiological activity) were analysed by seven laboratories participating in the study. The required detection limit was 1 mg kg^-1 (expressed in microbiological activity). For spiramycin, acceptable values for the sensitivity (at least 95%) indicating the rate of correct positive results were obtained for samples containing this substance at or above 2 mg kg^-1, whereas at 1 mg kg^-1, the sensitivity rate dropped to about 70%. Therefore, it was concluded that the detection limit was 2 mg kg^-1. For tylosin and virginiamycin, acceptable values of the sensitivity were obtained for all concentrations including 1 mg kg^-1. Therefore, the method fulfils the criterion regarding the required sensitivity at the target detection limit for tylosin and virginiamycin.     

Survey of ochratoxin A and deoxynivalenol in stored grains from the 1999 harvest in the UK

Three hundred and twenty samples from the 1999 UK harvest comprising wheat (201 samples), barley (106) and oats (13) were analysed for ochratoxin A and deoxynivalenol. A small number of organic samples was also obtained. Samples were collected from farms, central stores, mills, maltings and ports from across the UK from February to April 2000. Ochratoxin A and deoxynivalenol analysis was by affinity column clean up and high-performance liquid chromatography with fluorescence and ultraviolet light detection, respectively, with limits of detection of 0.2 and 20 μg kg^-1. The survey found ochratoxin A at below 5 μg kg^-1 in 97% of the samples indicating satisfactory storage conditions. The remaining 3% of the samples contained ochratoxin A at levels between 5.2 and 231 μg kg^-1, but none of these samples was intended for human consumption. Deoxynivalenol was detected in 88% of all samples, with 83% below 100 μg kg^-1; the maximum level was 600 μg kg^-1. Twenty samples containing deoxynivalenol at or above 150 μg kg^-1 by high-performance liquid chromatography were all confirmed by gas chromatography/mass spectrometry. Nivalenol was also detected by gas chromatography/mass spectrometry at levels of 50 μg kg^-1 or higher in 18 of 20 samples where deoxynivalenol was confirmed.     

Lead and cadmium in meat and meat products consumed by the population in Tenerife Island, Spain

The aim of this study was to determine the levels of lead and cadmium in chicken, pork, beef, lamb and turkey samples (both meat and meat products), collected in the island of Tenerife (Spain). Lead and cadmium were measured by graphite furnace atomic absorption spectrometry (GFAAS). Mean concentrations of lead and cadmium were 6.94 and 1.68 µg kg^-1 in chicken meat, 5.00 and 5.49 µg kg^-1 in pork meat, 1.91 and 1.90 µg kg^-1 in beef meat and 1.35 and 1.22 µg kg^-1 in lamb meat samples, respectively. Lead was below the detection limit in turkey samples and mean cadmium concentration was 5.49 µg kg^-1. Mean concentrations of lead and cadmium in chicken meat product samples were 3.16 and 4.15 µg kg^-1, 4.89 and 6.50 µg kg^-1 in pork meat product, 6.72 and 4.76 µg kg^-1 in beef meat product and 9.12 and 5.98 µg kg^-1 in turkey meat product samples, respectively. The percentage contribution of the two considered metals to provisional tolerable weekly intake (PTWI) was calculated for meat and meat products. Statistically significant differences were found for lead content in meats between the chicken and pork groups and the turkey and beef groups, whereas for cadmium concentrations in meats, significant differences were observed between the turkey and chicken, beef and lamb groups. In meat products, no clear differences were observed for lead and cadmium between the various groups.     

Determination of ochratoxin A in virgin olive oils of Greek origin by immunoaffinity column clean-up and high-performance liquid chromatography

An improved specific analytical method for ochratoxin A (OTA) determination in olive oil is described, using a methanolic-aqueous extraction, an immunoaffinity column clean up step and high-pressure liquid chromatography with fluorescence detection. The mean recovery was found at 108% (relative standard deviation, RSD = 4.7%) and the detection limit (DL) was estimated at 4.6 ng kg^-1. Along with OTA, aflatoxin B₁ (AFB₁) was determined using the same extract. The recovery factor was 84.8% (RSD = 17.8%) and the DL was 56 ng kg^-1 olive oil. Both determinations were applied in 50 samples of olive oil originated from representative regions of Greece. Results revealed the presence of OTA in 88% of samples tested (n = 44, mean 267 ng kg^-1). Among them, 10 were contaminated with more than 500 ng kg^-1 (median 568 ng kg^-1), 10 with 200-500 ng kg^-1 (median 260 ng kg^-1), 15 with 100-200 ng kg^-1 (median 140 ng kg^-1), nine with DL-100 (median 60 ng kg^-1) and in six samples, OTA was not detectable. Interestingly, most contaminated samples were from Southern Greece. Results of AFB₁ determination showed the presence of aflatoxin B₁ (60 ng kg^-1) in only one olive oil sample also from Southern Greece. The levels of OTA found in Greek olive oil were relatively low as compared with other commodities such as cereals or wine reported in the literature.     

Determination of selected mycotoxins in mould cheeses with liquid chromatography coupled to tandem with mass spectrometry

A simple and feasible method is described for analysing nine mycotoxins in cheese matrix. The method involves liquid extraction followed by high performance liquid chromatographic separation and mass spectrometric detection of the analytes, and allows the determination of aflatoxins B1, B2, G1, G2 and M1, ochratoxin A, mycophenolic acid, penicillic acid and roquefortine C simultaneously. Average recoveries of the mycotoxins from spiked samples at concentration levels of 5-200 µg kg^-1 ranged from 96-143%. Within-day relative standard deviations at these concentration levels varied from 2.3-12.1%. The limit of quantification for aflatoxin M1 was 0.6 µg kg^-1 and for the other compounds 5 µg kg^-1. The method developed was applied for analysing these mycotoxins in blue and white mould cheeses purchased from Finnish supermarkets. Roquefortine C was detected in all of the blue mould cheese samples in concentrations of 0.8-12 mg kg^-1. One blue cheese contained also 0.3 mg kg^-1 mycophenolic acid. The other investigated mycotoxins were absent in the samples.