Nathan Rummel

Determination and Confirmation of Melamine Residues in Catfish, Trout, Tilapia, Salmon, and Shrimp by Liquid Chromatography with Tandem Mass Spectrometry

Pet and food animal (hogs, chicken, and fish) feeds were recently found to be contaminated with melamine (MEL). A quantitative and confirmatory method is presented to determine MEL residues in edible tissues from fish fed this contaminant. Edible tissues were extracted with acidic acetonitrile, defatted with dichloromethane, and cleaned up using mixed-mode cation exchange solid-phase extraction cartridges. Extracts were analyzed by liquid chromatography with tandem mass spectrometry with hydrophilic interaction chromatography and electrospray ionization in positive ion mode. Fish and shrimp tissues were fortified with 10-500 microg/kg (ppb) of MEL with an average recovery of 63.8% (21.5% relative standard deviation, n = 121). Incurred fish tissues were generated by feeding fish up to 400 mg/kg of MEL or a combination of MEL and the related triazine cyanuric acid (CYA). MEL and CYA are known to form an insoluble complex in the kidneys, which may lead to renal failure. Fifty-five treated catfish, trout, tilapia, and salmon were analyzed after withdrawal times of 1-14 days. MEL residues were found in edible tissues from all of the fish with concentrations ranging from 0.011 to 210 mg/kg (ppm). Incurred shrimp and a survey of market seafood products were also analyzed as part of this study.

Evaluation of the renal effects of experimental feeding of melamine and cyanuric acid to fish and pigs.

OBJECTIVE To determine whether renal crystals can be experimentally induced in animals fed melamine or the related triazine compound cyanuric acid, separately or in combination, and to compare experimentally induced crystals with those from a cat with triazine-related renal failure. ANIMALS 75 fish (21 tilapia, 24 rainbow trout, 15 channel catfish, and 15 Atlantic salmon), 4 pigs, and 1 cat that was euthanatized because of renal failure. PROCEDURES Fish and pigs were fed a target dosage of melamine (400 mg/kg), cyanuric acid (400 mg/kg), or melamine and cyanuric acid (400 mg of each compound/kg) daily for 3 days and were euthanatized 1, 3, 6, 10, or 14 days after administration ceased. Fresh, frozen, and formalin-fixed kidneys were examined for crystals. Edible tissues were collected for residue analysis. Crystals were examined for composition via Raman spectroscopy and hydrophilic-interaction liquid chromatography-tandem mass spectrometry. RESULTS All animals fed the combination of melamine and cyanuric acid developed goldbrown renal crystals arranged in radial spheres (spherulites), similar to those detected in the cat. Spectral analyses of crystals from the cat, pigs, and fish were consistent with melamine-cyanurate complex crystals. Melamine and cyanuric acid residues were identified in edible tissues of fish. CONCLUSIONS AND CLINICAL RELEVANCE Although melamine and cyanuric acid appeared to have low toxicity when administered separately, they induced extensive renal crystal formation when administered together. The subsequent renal failure may be similar to acute uric acid nephropathy in humans, in which crystal spherulites obstruct renal tubules.

Residue depletion of melamine and cyanuric acid in catfish and rainbow trout following oral administration

The intentional addition of triazines such as melamine to animal feeds and the lack of information about residue accumulation in food animals caused global concerns for food safety during 2007 and 2008. We report the results of a good laboratory practices (GLP) study to determine melamine and cyanuric acid residues in catfish and trout filets harvested at 1, 3, 7, 14, 28, and 42 days after a single oral dose of 20 mg/kg body weight of melamine, cyanuric acid, or melamine and cyanuric acid together. Peak melamine concentrations were 12.73 mg/kg (ppm) in catfish (mean = 9.98), 12.26 mg/kg in trout (mean = 7.89) on day 1. Within 7 days (catfish) or 14 days (trout) residues were <2.5 mg/kg, a level in foods accepted by many risk assessors worldwide to be unlikely to pose health risks to consumers. Peak cyanuric acid residues also occurred on day 1, 0.68 mg/kg in catfish (mean = 0.46), 2.59 mg/kg in trout (mean = 0.86). Cyanuric acid muscle residues were <2.5 mg/kg by day 3. The half-lives for melamine and cyanuric acid ranged between 1 and 4 days. Renal crystals formed in fish given both melamine and cyanuric acid, persisting for weeks after the single dose.

DETERMINATION OF SULFAMETHAZINE AND ITS MAJOR METABOLITES IN EGG ALBUMIN AND EGG YOLK BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A quantitative liquid chromatographic method for the determination of sulfamethazine (SMZ), N4-acetyl sulfa-methazine (N4-acetyl-SMZ), and desamino sulfamethazine (desamino-SMZ) in egg albumin and egg yolk is described. Egg albumin or yolk was homogenized in acetonitrile and centrifuged. The supernatant was evaporated to dryness and residue reconstituted in the mobile phase. Albumin extract was directly analyzed by high performance liquid chromatography (HPLC). Hexane was added to the yolk sample, vortex mixed, and centrifuged to separate the layers. The top hexane layer was removed and a small amount of salt was added to break the emulsions. The lower aqueous layer was analyzed by HPLC. The HPLC system included a reversed phase column, a gradient mobile phase of 5–15% acetonitrile and 0.01M phosphate buffer, and a UV detector set at 268 nm. The recovery of SMZ and N4-acetyl-SMZ, both fortified at 1 ppm levels, from egg albumin was 101 and 88% and from egg yolk was 79 and 91%, respectively. The recovery o...

Determination of trichlormethiazide in bovine milk by high-performance liquid chromatography

A liquid chromatographic procedure was developed and validated for the quantitative determination of trichlormethiazide (TCMTZ) in bovine milk. Whole milk was defatted by initial centrifugation at 4 degrees C. The resulting skim milk was treated with lead acetate and acetonitrile, vortex mixed, and centrifuged. The acetonitrile from the supernatant was back extracted in ethyl acetate. The organic solvent mixture which contained TCMTZ was further treated with sodium tungstate, vortex mixed, and centrifuged. The top organic layer was removed and evaporated to dryness; the resulting residue was reconstituted in the mobile phase, and the final extract was analyzed by high-performance liquid chromatography (HPLC). The HPLC conditions employed included a polymer column, a mobile phase consisting of 30% acetonitrile or 30% acetonitrile-tetrahydrofuran (2:1, v/v) in a phosphate buffer (pH 3), and a UV detection at 225 nm. The average recoveries of TCMTZ from milk fortified at 7, 14, 35, 70, and 140 ppb were 88, 93, 117, 110, and 99%, respectively, with corresponding C.V. values of 7, 18, 11, 9, and 21%. The method was validated by assaying milk obtained from a cow dosed with Naquasone. TCMTZ concentration was detected only in the 8 h post dose milk samples and was determined to be 6 ppb.

Residue Depletion of Tritium-Labeled Ivermectin in the Muscle Tissues of Aquacultured Atlantic Salmon, Tilapia, and Catfish following Oral Treatment

Abstract The residue depletion and metabolism profiles of tritium-labeled ivermectin in the muscle tissues of aquacultured Atlantic salmon Salmo salar, hybrid tilapia (Nile tilapia Oreochromis niloticus × Mozambique tilapia O. mossambicus; hereafter, “tilapia”) and channel catfish Ictalurus punctatus following oral treatment are reported. Fish were administered 3H-ivermectin at the dose level of 0.1 mg/kg of body weight (9–10 μ curies [Ci]) in a gel capsule via a stomach tube. At each postdose withdrawal time (1, 7, 14, 21, and 28 d), six fish of each species were sedated, euthanized, bled, and scaled, and fillets with adhering skin (except for catfish) were collected. The muscle fillets were homogenized in dry ice and an aliquot of each was extracted in acetonitrile followed by a high-performance liquid chromatographic analysis to determine the presence of parent ivermectin and its potential metabolites. The analysis of the muscle extracts revealed that the parent 3H-ivermectin is the major residue prese...

Marker Residue Determination of Tritium-Labeled Ivermectin in the Muscle of Aquacultured Largemouth Bass, Hybrid Striped Bass, and Yellow Perch following Oral Treatment

The residue depletion profiles of tritium-labeled ivermectin and its metabolites in the muscle of aquacultured largemouth bass (LMB), hybrid striped bass (HSB), and yellow perch (YP) following oral treatment are reported. Fish were administered ³H-ivermectin at the dose level of 0.1 mg/kg body weight (7-9 μCi) in a gel capsule via stomach tube. At each postdose withdrawal time, six fish of each species were sedated with buffered MS-222 and blood samples taken. Fish were then euthanized, and fillets with adhering skin (scales removed) and bile samples were collected. The muscle fillets were homogenized in dry ice to a fine powder. Aliquots of tissue, plasma, and bile were assayed for total radioactive residue (TRR). The homogenized muscle was extracted in acetonitrile or methanol followed by high-performance liquid chromatographic (HPLC) analysis to determine the presence of parent ivermectin and its potential metabolites. The highest TRR concentrations (ivermectin equivalents) of 53, 45, and 44 ng/g (ppb) were obtained on postdose day 1 for HSB, LMB, and YP, respectively. The TRR depleted most slowly in HSB to 25 ppb at day 91, followed by YP to 19 ppb at day 42 and then by LMB to 22 ppb at day 35. The total residue of ivermectin and its metabolites by HPLC analysis followed the same depletion pattern in the three species. Additionally, the depletion rate of TRR of ³H-ivermectin in the three species followed the pattern bile > plasma > muscle. The results further indicate that one of the polar metabolites of ivermectin could serve as a potential marker residue as an indication of use, rather than the parent ivermectin.