Development and validation of a new method for determining nitrofuran metabolites in bovine urine using liquid chromatography - tandem mass spectrometry.

BACKGROUND: The use of nitrofurans as veterinary drugs in food-producing animals is banned throughout the European Union. Nevertheless, nitrofuran metabolites have been detected not only in animal products, but also in bovine urine. At present there are no methods yet published for the simultaneous detection of nitrofuran metabolites in bovine urine. OBJECTIVES: To develop and validate a method for determination of four key nitrofuran metabolites in bovine urine. MATERIAL AND METHODS: The four nitrofuran metabolites (nitrofurantoin, furazolidone, nitrofurazone and furaltadone), were determined in bovine urine using LC-ESI-MS/MS. The procedure required an acid-catalysed release of protein-bound metabolites, followed by their in situ conversion into 2-nitrobenzaldehyde (NBA) derivatives. The sample clean-up was performed using a polymer extraction cartridge before hydrolysis. Nitrofuran metabolites were then determined using electrospray ionization in the positive mode, that had previously been separated on a Phenomenex Luna C-18 column. RESULTS: The method was validated in accordance with the procedure outlined in the Commission Decision No. 2002/657/ EC. Urine samples were spiked with nitrofuran metabolite solutions at levels of 0.5, 1.0, 1.5 and 2.0 microg/kg. Recoveries ranged between 90 - 108% (inter standard-corrected), with a repeatability precision (RSD) of less than 19% for all four analytes. The decision limit (CC) and detection capability (DC) were obtained from a calibration curve and lay respectively within the following ranges: 0.11 - 0.34 microg/kg and 0.13 - 0.43 microg/kg. CONCLUSIONS: The developed and validated LC-ESI-MS/MS method allows four nitrofuran metabolites to be identified and quantitated in bovine urine. This analytical procedure meets the criteria defined in the Commission Decision No. 2002/657/EC.

Analysis of 5-nitrofuran derivatives in biological objects.

Original methods for identification and quantitative photometry of 5-nitrofurane derivatives based on the use of known rhodanine reagents were developed and used in clinical analysis.

Renal metabolism of formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl]-hydrazide.

Formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl]-hydrazide (FNT) is a potent renal carcinogen in the rat. This study assessed the metabolism of FNT by the isolated perfused rat kidney and whole rat. The glomerular filtration rate and the fractional excretion of sodium for the isolated perfused kidney indicated that under the conditions of these experiments FNT did not alter these renal parameters. The half-life (t1/2) for FNT in the isolated perfused kidney was 67 +/- 8 min. Using HPLC, a metabolite of FNT was observed in urine from the isolated perfused kidney. This metabolite had absorbance at 385 nm but not 254 nm and could not be detected electrochemically at +500 mV. While the excretion of FNT decreased with time of perfusion, the metabolite excretion increased. Whole animal studies demonstrated that FNT is rapidly cleared from blood within the first 5 min of administration. The FNT metabolite was excreted at approximately the same rate from 0-30 and 30-60 min after FNT administration. The metabolite was not observed in media from FNT perfused kidneys or plasma from animals administered FNT. Analysis of purified metabolite by liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS) determined the structure to be 5-nitro-2-furonitrile. This structure assignment was verified by chemical synthesis. Results demonstrate target organ metabolism of carcinogen.

Some pharmacokinetic data about furaltadone and nitrofurazone administered orally to preruminant calves.

A single oral dosage of furaltadone and nitrofurazone (14.0 mg/kg) to 5 preruminant calves (in a cross-over trial) revealed mean maximum plasma concentration of 2.5 and 3.5 microgram/ml, respectively, at approximately 3 h after administration. The final elimination half-lives of furaltadone and nitrofurazone were 2.5 and 5 h, respectively. Urinary recovery of these two nitrofurans in 3 calves revealed approximately 2% of the orally administered dose. The renal clearance of the unbound drugs did not differ (for both drugs approximately 0.42 ml/min/kg); furaltadone clearance was strongly related to urine flow.

Comparative physiological disposition of two nitrofuran anti-microbial agents.

The physiological disposition of two nitrofuran derivatives used as antimicrobial agents for the treatment of acute infectious diarrhoea was evaluated in humans and animals. Upon administration of a single oral dose (600 mg) of nifurzide or nifuroxazide, no unchanged parent drug was detected in human blood or urine. In rats given 14C-nifurzide and 14C-nifuroxazide at a dose of 10 mg kg-1, 5 per cent and 17 per cent of the dose of nifurzide and nifuroxazide, respectively, were excreted in urine over a 48-hour period. None of this radioactivity was present as unchanged drug, indicating that renal excretion of both drugs occurs as metabolites. In the faeces 20 per cent of the radioactivity recovered was associated with unchanged nifuroxazide as compared with 100 per cent for nifurzide. Whole body autoradiography using rats showed that after oral administration of 14C-nifurzide and 14C-nifuroxazide, most of the radioactivity remained in the gastrointestinal lumen.

Main excretion metabolites of 7-methoxy-2-nitronaphtho[2,1-b]furan (R 7000) in rats.

Major metabolites, isolated from rat bile and urine after administration of a single dose of 7-methoxy-2-nitronaphtho[2,1-b]furan (R 7000; MNNF) labelled with 14C on the furan ring and on the methoxy group, were identified by comparison of their chromatographic behaviour and mass spectra with synthetic authentic reference compounds. Analysis of metabolites indicated three metabolic pathways for this compound in vivo, namely, demethylation of the methoxy group, hydroxylation of the aromatic ring and cleavage of the furan ring, followed by the reduction of the nitro group to amine.

In vivo and in vitro pharmacokinetics and fate of furaltadone in meat- and milk-producing animals.

The metabolism of furaltadone was examined by an in vitro hepatic study in cows and goats and an in vivo study in goats using 14C-labeled and unlabeled drug. The half-life of furaltadone was 13 min in the homogenates of caprine and bovine liver and 35 min in the in vivo study of the goat. Less than 2% of the parent drug was present in the urine of animals dosed either intravenously or intramammarily. No furaltadone was detected in the milk after 24 h. Overall, the parent compound was rapidly absorbed, distributed, and widely degraded in the lactating goat. The compound, labeled at the 2-formyl carbon of the furan ring, had a radioactivity recovery of 81% in the feces and urine. Of the total radioactivity, 99.4% infused into the udder had been absorbed after 72 h. Tissue distributions of radioactivity in decreasing order of abundance were: kidney, udder, liver, duodenum, muscular tissue, adipose tissue, and bile.

Mutagenicity studies with nitrofurans. III. Mutagenicity testing of nitrofurylacrylic acid in human blood and urine.

Human blood and urine mutagenicity of 3-(5-nitro-2-furyl)acrylic acid (5-NFA) was analysed by using Salmonella typhimurium indicator strains TA100 AND TA98 and the cytogenetic analysis of human peripheral lymphocytes. 8 human volunteers were given doses of 1 g 5-NFA per os. The mutagenic effect in blood was analysed after 0, 0.5, 1, 2 and 4 h, in urine after 0, 2 and 4 h. Cytogenetic analysis was done 0, 24 and 72 h after administration of 5-NFA. The experiment was repeated with 3 volunteers in the course of 96 h. When each of 8 volunteers consumed 1 g of 5-NFA, the mutagenicity was observed in 6 blood samples 1 h after exposure for strain TA98 (doubled number or revertants) and in all urine samples taken between the 2nd and 6th hours for both strains used. 7 volunteers given 10 mg 5-NFA in wine (2 sets) showed no mutagenicity of blood or urine for TA100 or TA98 indicator strains. These results are believed to indicate an enhanced elimination of 5-NFA from the human body.

Mutagenic activity of carcinogenic and noncarcinogenic nitrofurans and of urine of rats fed these compounds.

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT greater than nitrofurantoin greater than 5-nitro-2-furamidoxime greater than 5-nitrofurfurylidene diacetate greater than 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with beta-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.

A simple method for detection and analysis of carcinogenic nitrofuran compounds and their metabolites by combining chromatography and spot mutation tests.

A simple, sensitive, and convenient method combining thin-layer chromatography and a spot test for mutagenicity of Salmonella typhimurium TA100 was utilized for the analysis of urine of rats fed N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT), a potent experimental urinary bladder carcinogen. Three metabolites of FANFT were detected in urine, and one of these, accounting for 33% of the urinary metabolites of FANFT, was identified as 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT). ANFT was the only urinary metabolite that clearly demonstrated mutagenic activity, suggesting that ANFT may be a proximate vesical carcinogen of FANFT.

Metabolic and photochemical hydroxylation of 5-nitro-2-furancarboxaldehyde derivatives.

The potassium salt of 1-[[(5-aco-nitro-4,5-dihydro-4-oxo-2-furanyl)methylene]amino]-2,4-imidazolidinedione (4) was isolated from the urine of rats fed nitrofurantoin. An aldehyde absorbing at 400 nm was synthesized photochemically, in less than 1% yield, from 5-nitro-2-furancarboxaldehyde diacetate (1), and the hydroxylamine (2), 3-amino-2-oxazolidinone (3a-c), and 1-amino-2,4-imidazolidinedione (4) derivatives were prepared. On the basis of ir and NMR data 2, 3b,c, and 4 are considered derivatives of 4-hydroxy-5-nitro-2-furancarboxaldehyde which are mainly in the aci-nitro form. Methyl and ethyl nitronic esters of 3b were synthesized. The photochemical hydroxylation of 1 also yields 3,4-dihydroxy-5-nitro-2-furancarboxaldehyde, isolated as 3-[[(3,4-dimethoxy-5-nitro-2-furanyl)-methylene[amino[-2-oxazolidinone (7).

Effects of different drugs on the urinary excretion of nifurpipone (NP) and of nitrofurantoin (NTF) in the rat.

A study was made on possible interferences of several drugs on the urinary excretion of nifurpipone and of nitrofurantoin orally administered to rats. Phenobarbital, a microsomial enzyme inducer, SKF 525 A, a microsomial enzyme inhibitor, probenecid, an inhibitor of the tubular acid secretory system, quanine, an inhibitor of the tubular alkaline secretory system, and hydrochlorothiazide, a diuretic, were studied. None of these treatments altered in an important way the urinary excretion of nifurpipone or of nitrofurantoin.

1(((5-Nitrofuranyl)methylene)amino)-4 and/or-5-substituted 2-imidazolidinones.

A series of 1-[[(5-nitrofuranyl)methylene]amino]-4- and/or -5-substituted 2-imidazolidinones was prepared utilizing three different reaction sequences. The structure of 4, the product derived from 4-methyl-2-imidazolidinone (2a), was verified by synthesis using an alternate, unequivocal route. The levo isomer l-4 was prepared by a series of reactions starting with L(+)-2-amino-1-propanol (l-10). All of the nitrofurans were examined for potential use as chemotherapeutic agents for urinary tract infections. Based on the high level of activity in the urine and the in vitro antibacterial activity (MIC) 4, l-4, and 16 are considered to be the most active as urinary tract agents.