Automated determination of oxytetracycline residues in muscle, liver, milk and egg by on-line dialysis and post-column reaction detection HPLC.

An automated method for control of oxytetracycline (OTC) residues in chicken and bovine muscle, salmon liver, bovine milk and hen egg has been developed. Tissue homogenate, decreamed milk or whole egg solution was dialysed and the dialysate enriched on a small polystyrene column on-line to HPLC. OTC and the internal standard (tetracycline) were separated on a polystyrene column by ion-pair chromatography. The column effluent was mixed with sodium hydroxide and irradiated at 366 nm. Monitoring the resulting derivatives with a fluorescence detector (excitation: 358 nm, emission: 460 nm), OTC could be detected at 1 ng/ml in milk, 1 ng/g in egg, 3-4 ng/g in muscle and 8 ng/g in liver. Relative standard deviations at 50 and 200 ng/g (milk: 20 and 100 ng/ml) ranged from 1.6 to 3.1%.     

Residues of lindane in plasma and fat of sheep after dipping

Ten 5-6 year-old unshorn ewes were dipped in a 0.025% lindane solution. Lindane residues in fat and plasma were analysed by gas liquid chromatography with an electron capture detector. The sensitivity of the method was 0.02 microgram/g for fat and 0.005 microgram/ml for plasma. Blood samples of 5 sheep were collected up to 84 days. The highest concentration of lindane detected in plasma was 107 +/- 37 ng/ml, 48 h after treatment. The disposition of lindane in plasma was studied by linear regression. Omental fat samples were removed by biopsies at 7 or 14 day intervals for 154 days. Fat concentrations of lindane were very high, respectively 30.46 +/- 14.6 ug/g, 8.09 +/- 4.57 micrograms/g, and 1.72 +/- 0.81 micrograms/g, 7, 28 and 70 days after dipping. In plasma and fat, the coefficients of elimination calculated by linear regression were similar (0.00190 +/- 0.00053 h-1 for plasma, 0.00196 +/- 0.00031 h-1 for fat). The tolerance recommended in France (2 micrograms/g in fat) was obtained 59.7 +/- 9.7 days after this treatment.     

Histological Changes in Collagen Related to Textural Development of Prawn Meat During Heat Processing

Heat processing enhanced firmness and degree of shrinkage deformation of kuruma prawn Penaeus japonicus meat. In histological experiments, most collagen fiber in perimysium, epimysium, and subcuticular connective tissue maintained structures after heat-processing in hot water at 70°C for 30 min. Crude collagen fiber fractions (residues after alkali extraction, RS-AL) prepared from the muscle were measured for hot-water solubility and compared with the RS-AL from the muscle of carp Cyprinus carpio. Collagen in the RS-AL of prawn muscle had very low hot-water solubility (about 23.5% at 70°C) compared with that of carp. These results suggested that collagen had important functions in promoting thermal shrinkage and hardening of prawn meat and in maintaining mechanical strength of heat-processed meat.     

Occurrence and density of vibrio parahaemolyticus in live edible crustaceans from markets in China

Vibrio parahaemolyticus is a marine bacterium causing foodborne disease. Occurrence of the bacterium was investigated in six species of edible crustaceans available from markets in mainland China. The bacterium was detected in 22 of 45 whole-body, shell, and feces samples, including mitten crabs, which are supposed to be produced in freshwater ponds. The mean densities ranged from 2.8 log CFU/g in mitten crabs (Eriocheir sinensis) to 5.1 CFU/g in giant tiger prawns (Penaeus monodon). In hemolymph and muscle samples collected axenically, V. parahaemolyticus was detected in all of the prawns at a mean density of 2.6 log most probable number (MPN)/g, in two of five striped stone crabs (Charybdis feriatus) at a mean density of 1.1 log MPN/ml, and two of five mangrove mud crabs (Scylla serrata) at a mean density of 1.3 log MPN/ml. When six mitten crabs were collected from two freshwater ponds in China and were examined, V. parahaemolyticus was not detected. It seemed that cross-contamination occurred among live crustaceans at the markets. The results suggest that proper handling, storage, and cooking of these crustaceans will be necessary to lessen the risk of foodborne illness from V. parahaemolyticus.     

Residues of doxycycline and oxytetracycline in eggs after medication via drinking water to laying hens

Doxycycline (DOTC) and oxytetracycline (OTC) were dissolved in drinking water (0.5 g/l) and supplied to laying hens for 7 consecutive days. Eggs laid were collected daily during and after medication, and the antibiotic concentrations in the yolk and albumin were determined by the cup-plate method with Bacillus cereus var. mycoides ATCC 11778. The concentrations of both antibiotics were increased in yolk day by day with the advance in medication, reached peaks 2 days after withdrawal and then declined gradually. Mean peak concentrations in the yolk were 6.70 micrograms/g for DOTC and 1.42 micrograms/g for OTC. Peak concentrations in the albumin occurred in the middle stage of medication, where the mean values were 12.24 micrograms/g for DOTC and 1.03 micrograms/g for OTC. DOTC was detected in albumin until 24 days after withdrawal and for 2 days more in yolk than in albumin. OTC was detected in yolk until 9 days after withdrawal. The depletion period of OTC was shorter for the albumin, where the residue disappeared in all eggs 6 days after withdrawal. In spite of similarities between DOTC and OTC in structure, DOTC was deposited in higher concentrations and lasted for a longer period in eggs. This characteristic was considered due to its greater lipophilicity, closely correlated with oral absorption and tissue penetration.     

Determination of 4-hexylresorcinol residues in prawns and crabs

A method for the determination of 4-hexylresorcinol residues in prawn and crab meat by HPLC was developed. 4-Hexylresorcinol in prawn and crab meats was extracted with methanol using a homogenizer. The extract was diluted 4 times with water, and the diluted solution was passed through a C18 cartridge. The cartridge was washed with water and methanol-water (4 : 6), and then 4-hexylresorcinol was eluted with acetonitrile-0.1% phosphoric acid (55 : 45). The eluate was separated on a Capcell Pak C18 MG column with a mobile phase of acetonitrile-0.1% phosphoric acid (6 : 4) and 4-hexylresorcinol was determined with a UV detector (210 nm). Recoveries of 4-hexylresorcinol from commercial prawn and crab meats spiked at 1.0 and 10 microg/g were 82.4-92.2 and 88.9-91.8%, respectively. The determination limit of 4-hexylresorcinol was 1.0 microg/g in the samples.     

Effects of temperature on the elimination of benzocaine and acetylated benzocaine residues from the edible fillet of rainbow trout (Oncorhynchus mykiss)

The effect of temperature (7 degrees C and 16 degrees C) on the extent of accumulation and the elimination of benzocaine (BNZ) and its metabolite, acetylated benzocaine (AcBNZ), in the fillet tissue of rainbow trout was investigated. Residues were measured after bath exposure to an anesthetizing concentration of benzocaine (30 mg/l for 5 min) followed by a maintenance concentration (15 mg/l for 30 min). Immediately after exposure, the BNZ concentration in fillet tissue was approximately 27 micrograms/g at both temperatures; AcBNZ was 0.3 microgram/g at 7 degrees C and 0.6 microgram/g at 16 degrees C. The rates for elimination (alpha and beta) of BNZ and AcBNZ were not significantly different between the two temperatures. Terminal half-lives of elimination for BNZ were 1.62 h at 7 degrees C and 1.63 h at 16 degrees C; half-lives for AcBNZ were 2.36 h at 7 degrees C and 2.77 h at 16 degrees C.     

Postharvest Microbiology of Farm-reared, Tropical Freshwater Prawn (Macrobrachium Rosenbergii)

ABSTRACT: The microbiological quality of farm-reared, tropical freshwater prawn ( Macrobrachium rosenbergii ) stored at 2 different temperatures was studied. The prawn muscle was found to have the initial bacterial load of 10⁴ cfu/g. The lactics and vibrios were in the range of 10² cfu/g, while the E. coli , aeromonads, staphylococci, anaerobes, and molds were in the level of 10¹ cfu/g. Salmonella and Vibrio cholerae were present in the prawn muscle. The prawn muscle held at room temperature (28 ± 2 °C) was organoleptically acceptable up to 8 h, when the bacterial load was more than 10⁶ cfu/g. However, the prawn muscle stored at freezer temperatures (−10 to −15 °C) was found to be in acceptable condition even after 30 d of storage and the bacterial load was fluctuating in the range of 10³ to 10⁴ cfu/g.     

Determination of residues of oxolinic acid and flumequine in freeze-dried salmon muscle and skin by HPLC with fluorescence detection

A procedure for the determination of residues of oxolinic acid (OA) and flumequine (FLU) in freeze-dried salmon muscle with attached skin, using reversed-phase high-performance liquid chromatography, is described. OA and FLU were extracted by a solid-liquid extraction procedure: after addition of hydrochloric acid, extraction used successively ethyl acetate, sodium hydroxide and chloroform. Liquid chromatography was performed on a 5 μm PuroSpher RP-18E ® cartridge using acetonitrile and 0.02 M aqueous orthophosphoric acid solution as mobile phase, with fluorescence detection. The performance of the method was established by spiking tissues with OA and FLU before the freeze-drying step. The method was linear over the concentration range 50-2000 ng/g freeze-dried tissue. Limits of detection and quantitation were 3.2 and 16 ng/g wet weight tissue respectively both for OA and FLU. Mean extraction recoveries of OA and FLU from freeze-dried tissue were 85.5 and 85.2% respectively. The method is suitable as a regulatory one for determination of residues of OA and FLU in freeze-dried salmon tissue.     

Retention of Oxytetracycline Residues in Cooked Channel Catfish Fillets

Catfish (mean body weight 0.87 kg) treated with oxytetracycline (OTC) at 150.0 mg/kg body weight for 10 days and slaughtered 18 hr after last feeding had higher OTC residue levels than those receiving OTC at 37.5 or 75.0 mg/kg. Individual differences in feed uptake and metabolism of OTC among catfish might contribute to variability in residue. Baking and smoking at 190°C were more effective in reducing OTC residues than frying. The reduction appeared to be related to the final temperature reached and duration of cooking. Results confirmed that common cooking procedures may not completely degrade OTC in catfish fillets.     

Improvement of Red Color Development on the Surface of Kuruma Prawn Marsupenaeus japonicus under Various Conditions

The degree of red color development on the surface of prawns by cooking is an important index for food quality. In this study, we tested several factors that are thought to influence the red color development to identify possible correlations with various conditions. Live kuruma prawns, Marsupenaeus japonicus, (15.4 cm, 25.2 g on average) were used in this study. In case of cooking at 100 °C for 1 min after 24 h of storage at 0 °C, 5 °C, and 20 °C, the red color development rate of prawns stored at 5 °C and 20 °C was significantly lower than that of prawns cooked just after killing. In case of cooking at 100 °C, 80 °C, and 60 °C after storage for 24 h at 0 °C, there was no color development at 60 °C and significantly less color development at 80 °C compared to cooking just after killing. Preparation using 1% sodium carbonate before cooking at 80 °C could compensate for the lack of red color development. Short exposure of live kuruma prawns to low‐oxygen conditions had no influence on the color development, but putting the prawns in freshwater for 3 h significantly reduced the red color development rate. In conclusion, the storage time has little influence on the red color development when the cooking temperature is sufficiently high. However, in case a large amount of prawns is cooked followed by lowering the cooking temperature and/or prawns are exposed to serious stresses before cooking, an alkaline preparation could compensate for the lack of red color development.     

Tetrahydro-beta-carboline-3-carboxylic acid compounds in fish and meat: possible precursors of co-mutagenic beta-carbolines norharman and harman in cooked foods

The presence of tetrahydro-beta-carbolines and beta-carbolines was studied in raw, cooked and smoked fish and meat. 1,2,3,4-Tetrahydro-beta-carboline-3-carboxylic acid (THCA) usually was the major beta-carboline found, whereas 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA) appeared in smoked and 'well done' cooked samples. THCA was detected in raw fish (nd-2.52 micrograms/g), cooked fish (nd-6.43 micrograms/g), cooked meats (nd-0.036 microgram/g), smoked fish (0.19-0.67 microgram/g) and smoked meats (0.02-1.1 micrograms/g). Smoked and cooked samples contained higher amounts of THCA and MTCA than raw products. Deep cooking of fish and meat increased both THCA and MTCA, and this was accompanied by the formation of more beta-carbolines, norharman and harman. The tetrahydro-beta-carbolines THCA and MTCA were chemical precursors of the co-mutagens norharman and harman during cooking. These and previous results confirm that foods are an important source of beta-carbolines in humans.     

Excretion of tetracycline and chlortetracycline in eggs after oral medication of laying hens

After dosing laying hens orally with tetracycline (TC) through either drinking water (0.25 and 0.5 g/l for 5 days) or feed (300 and 600 ppm for 7 days), and chlortetracycline (CTC) through feed (600 ppm) residues were determined by an agar plate diffusion technique in cylinders with Bacillus cereus as test-organism, separately for albumen and for yolk. The sensitivity threshold was 0.07 micrograms/g in albumen and 0.15 micrograms/g in yolk for TC and 0.01 micrograms/g in albumen and 0.06 micrograms/g in yolk for CTC. Drug excretion via egg was 3-fold higher for TC than for CTC. The drug was excreted preferentially into the yolk (about 75% of the total amount) and the elimination period lasted between 6 and 11 days for TC and 9 days for CTC, after treatment. Tetracycline use in laying hens is discussed, taking into consideration the proposals presented by the joint FAO/WHO Expert Committee on Food Additives.     

Inhibitory effect of cysteine and glutathione on phenoloxidase from kuruma prawn (Penaeus japonicus)

The inhibitory effect of cysteine and glutathione on phenoloxidase (PO) from kuruma prawn were investigated. Cysteine and glutathione inhibited the oxidation of 3-(3,4-dihydroxylphenyl)-l-alanine (l-DOPA) catalyzed by kuruma prawn PO. Those thiol compounds showed competitive inhibition with K_i values of 0.45–0.46 mM. The inactivated PO could be partially recovered by the addition of copper acetate (0.01–0.2 mM). Almost complete restoration of PO activity was achieved with the addition of N-ethylmaleimide (NEM) in the range 0.05–0.2 mM, suggesting the importance of sulfhydryl groups of cysteine and glutathione for their inhibitory activity. Additionally, both cysteine and glutathione prevented the colour development by trapping the colour intermediates or reducing o-quinone to colourless compounds.     

Stability during cooking of anthelmintic veterinary drug residues in beef

Anthelmintic drugs are widely used for treatment of parasitic worms in livestock, but little is known about the stability of their residues in food under conventional cooking conditions. As part of the European Commission-funded research project ProSafeBeef, cattle were medicated with commercially available anthelmintic preparations, comprising 11 active ingredients (corresponding to 21 marker residues). Incurred meat and liver were cooked by roasting (40 min at 190°C) or shallow frying (muscle 8-12 min, liver 14-19 min) in a domestic kitchen. Raw and cooked tissues and expressed juices were analysed using a novel multi-residue dispersive solid-phase extraction method (QuEChERS) coupled with ultra-performance liquid chromatography-tandem mass spectrometry. After correction for sample weight changes during cooking, no major losses were observed for residues of oxyclozanide, clorsulon, closantel, ivermectin, albendazole, mebendazole or fenbendazole. However, significant losses were observed for nitroxynil (78% in fried muscle, 96% in roast muscle), levamisole (11% in fried muscle, 42% in fried liver), rafoxanide (17% in fried muscle, 18% in roast muscle) and triclabendazole (23% in fried liver, 47% in roast muscle). Migration of residues from muscle into expressed cooking juices varied between drugs, constituting 0% to 17% (levamisole) of total residues remaining after cooking. With the exception of nitroxynil, residues of anthelmintic drugs were generally resistant to degradation during roasting and shallow frying. Conventional cooking cannot, therefore, be considered a safeguard against ingestion of residues of anthelmintic veterinary drugs in beef.     

Production of perfluorinated carboxylic acids (PFCAs) from the biotransformation of polyfluoroalkyl phosphate surfactants (PAPS): exploring routes of human contamination

Perfluorinated acids are detected in human blood world-wide, with increased levels observed in industrialized areas. The origin of this contamination is not well understood. A possible route of exposure, which has received little attention experimentally, is indirect exposure to perfluorinated acids through ingestion of chemicals applied to food contact paper packaging. The current investigation quantified the load of perfluorinated acids to Sprague-Dawley rats upon exposure to polyfluoroalkyl phosphate surfactants (PAPS), nonpolymeric fluorinated surfactants approved for application to food contact paper products. The animals were administered a single dose at 200 mg/kg by oral gavage of 8:2 fluorotelomer alcohol (8:2 FTOH) mono-phosphate (8:2 monoPAPS), or the corresponding di-phosphate (8:2 diPAPS), with blood taken over 15 days post-dosing to monitor uptake, biotransformation, and elimination. Upon completion of the time-course study the animals were redosed using an identical dosing procedure, with sacrifice and necropsy 24 h after the second dosing. Increased levels of perfluorooctanoic acid (PFOA), along with both 8:2 PAPS congeners, were observed in the blood of the dosed animals. In the 8:2 monoPAPS-dosed animals, 8:2 monoPAPS and PFOA blood concentrations peaked at 7900 +/- 1200 ng/g and 34 +/- 4 ng/g respectively. In the 8:2 diPAPS-dosed animals, 8:2 diPAPS peaked in concentration at 32 +/- 6 ng/g, and 8:2 monoPAPS and PFOA peaked at 900 +/- 200 ng/g and 3.8 +/- 0.3 ng/g, respectively. Several established polyfluorinated metabolites previously identified in 8:2 FTOH metabolism studies were also observed in the dosed animals. Consistent with other fluorinated contaminants, the tissue distributions showed increased levels of both PFOA and the 8:2 PAPS congeners in the liver relative to the other tissues measured. Previous investigations have found that PAPS can migrate into food from paper packaging. Here we link ingestion of PAPS with in vivo production of perfluorinated acids.     

Hemocyanin a Most Likely Inducer of Black Spots in Kuruma Prawn Penaeus japonicus During Storage

ABSTRACT: Hemocyanin (Hc) from plasma of kuruma prawn was characterized as a potent inducer of black spots during storage. An oxygen transporter, hemocyanin was converted into phenoloxidase (PO)-like enzyme (HdPO) with SDS treatment as well as prophenoloxidase isolated from hemocytes. Both enzymes showed similar biochemical properties. The isolated PO, however, was so unstable that its activity was drastically reduced when frozen and thawed at -25°C for less than a week, while HdPO completely retained its activity for more than a month, suggesting that HdPO is a key factor in black spot development in freeze-thawed prawn.     

Ivermectin residues in the edible tissues of swine and cattle: effect of cooking and toxicological evaluation

Young male pigs (25-40 kg bw) were treated experimentally with a single 0.4 mg/kg bw, s.c. dose of ivermectin (Ivomec vet. inj., MSD). The disappearance of the drug from the edible tissues 7-21 days after treatment was studied using a sensitive high-performance liquid chromatographic method. The highest residue levels were found at the injection site (up to 59 and 2.6 mg/kg 7 and 14 days post-injection, respectively). Among the other tissues studied, the residue levels 7 days post-injection showed the following order: liver (less than or equal to 50 micrograms/kg) greater than kidney (less than or equal to 25 micrograms/kg) greater than muscle (less than or equal to 20 micrograms/kg). After 21 days only traces of ivermectin (less than or equal to 2 micrograms/kg) could be detected in the muscle and other edible tissues, including the injection site. Similar residue concentrations were found in slaughterhouse material from sows therapeutically treated with ivermectin for parasite infestation. An ordinary culinary preparation of the minced beef muscle from a bull treated with ivermectin resulted in a 45% (boiling) or 50% (frying) decrease in the drug residue. Based on the known toxic effects of the drug and the results of the present and other residue studies, the suggested withdrawal time for Ivomec in edible tissues of swine and cattle is 21 and 28 days, respectively.     

Rapid depletion of marbofloxacin residues in rabbit after therapeutic treatment

Although rabbit meat production represents a very small percentage of the world meat market, this percentage has been growing continuously during the last 30 years. Rabbit is considered a minor food species, and therefore no drugs are specifically registered for this animal. This situation encourages rabbit farmers to make off-label use of antibacterial drugs authorized for food-producing animal species other than rabbits. In the present study, the distribution and elimination of the fluoroquinolone antibacterial agent marbofloxacin in rabbit muscle, liver, and kidney was investigated. Marbofloxacin was chosen as a representative of a new generation of antibacterial drugs active against most gram-positive and gram-negative bacteria and mycoplasms; it is well tolerated and has short elimination times in bovine and swine species. Rabbits were treated with marbofloxacin at 2 mg kg of body weight(-1) for 5 days. Residual concentrations in liver, kidney, and muscle tissues were determined posttreatment with high-performance liquid chromatography and fluorescence detection. Marbofloxacin was rapidly distributed and eliminated from rabbit tissues. Concentrations were higher in the liver and kidney than in muscle. However, 48 h after the end of treatment, marbofloxacin concentrations dropped below the maximum residue level fixed for this antibacterial drug in cattle and pigs. Considering the efficacy of marbofloxacin for the treatment of the most common rabbit diseases, its tolerability, and its short elimination time as verified in the present study, use of this antibacterial drug could be extended to therapeutic treatment of rabbits.     

Increased inactivation of ozone-treated Clostridium perfringens vegetative cells and spores on fabricated beef surfaces using mild heat

Ozone treatment of beef surfaces enhanced the effectiveness of cooking temperatures ranging from 45 to 75 degrees C against enterotoxin-producing strains of Clostridium perfringens. Vegetative cells on beef surfaces at an initial concentration of 5.59 +/- 0.17 log CFU/g were reduced significantly (P < 0.05) to 4.09 +/- 0.72 log CFU/g and 3.50 +/- 0.90 log CFU/g after combined treatments with aqueous ozone (5 ppm) and subsequent heating at 45 and 55 degrees C, respectively. Spores on the beef surface were likewise significantly reduced from an initial concentration of 2.94 +/- 0.37 log spores per g to 2.07 +/- 0.38 log spores per g and 1.70 +/- 0.37 log spores per g after the combined treatment with aqueous ozone (5 ppm) and subsequent heating at 55 and 75 degrees C, respectively. Fluorescent nucleic acid stains were used with confocal fluorescence microscopy to show that spores remaining attached to the meat were protected from treatment-specific injury. This study provides evidence for the decreased resistance of both vegetative cells and spores of C. perfringens with ozone treatment that is followed by heat treatment at temperatures that would not otherwise be as effective, thus lowering the requirements for cooking beef while maintaining a margin of safety.