Pirlimycin residue in bovine liver--a case of reverse metabolism.

Samples of liver obtained from twenty dairy cows treated with pirlimycin hydrochloride (Pirsue) by the intramammary route and slaughtered at five different time intervals out to 28 days were incubated at room temperature and at 37 degrees C and analyzed by two HPLC-MS methods to examine the metabolite profile of the residue and to establish the quantitative relationship of the residue components. The evidence from these experiments suggests that the metabolism of pirlimycin in postmortem bovine liver is somewhat reversible, where the concentration of parent pirlimycin increases in the incubated liver with a concomitant reduction in the concentration of the pirlimycin sulfoxide metabolite. This increased parent-drug residue phenomenon is limited to liver and was not observed in kidney or muscle. The highest relative change in concentration was observed for low level biologically incurred samples and appeared to be a saturable process following Michaelis-Menten kinetics. All of the evidence collected appears to indicate that the phenomenon is the result of residual enzyme activity present in the postmortem liver samples and likely involves some type of reductase enzyme capable of reducing sulfur-oxidized substrates to the sulfide state. No attempts were made to identify specific enzymes responsible for this phenomenon.

Identification and determination of pirlimycin residue in bovine milk and liver by high-performance liquid chromatography-thermospray mass spectrometry.

Determinative and confirmatory methods of analysis for pirlimycin (I) residue in bovine milk and liver have been developed based on HPLC-thermospray (TSP) MS. Milk sample preparation consisted of precipitating the mill proteins with acidified acetonitrile followed by a solvent partitioning with a mixture of n-butyl chloride and hexane extraction of I from the aqueous phase into methylene chloride (MC), and solid-phase extraction clean-up. For liver, samples (2 g) were extracted with 0.25% trifluoroacetic acid in acetonitrile. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, then extracted with MC. The MC was evaporated to dryness and the dried residue reconstituted in 2.0 ml of 0.1 M ammonium acetate for analysis. A chromatographically resolved stereoisomer of I with TSP-MS response characteristics identical to I was used as an internal standard (I.S.) for quantitative analysis based on the ratio of peak areas of I to I.S. in the protonated molecular-ion chromatogram at m/z 411.2. The method for milk was validated by the analysis of control milk samples spiked with I at concentrations from 0.05 to 0.8 micrograms/ml. The overall recovery of pirlimycin across this concentration range was 95.4% +/- 8.7%. The limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were validated to be 0.05 micrograms/ml and 0.10 micrograms/ml, respectively. The method for liver was validated by the analysis of control liver samples spiked with I at concentrations ranging from 0.025 to 1.0 micrograms/g. The overall recovery of pirlimycin was 97.6% +/- 5.1% in this concentration range. The validated limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were 0.025 micrograms/g and 0.10 micrograms/g, respectively. Four diagnostic ions for I were monitored for confirmation: the pseudo-molecular ions (M+H)+ at m/z 411.2 (35Cl) and m/z 413.2 (37Cl), and fragment ions at m/z 375.2 and 158.1. Confirmatory criteria were defined for these assays.

Determination of ceftiofur and its desfuroylceftiofur-related metabolites in swine tissues by high-performance liquid chromatography.

An HPLC method was developed and validated for the determination of ceftiofur-related metabolites that have the potential to be microbiologically active in swine muscle, kidney, liver and fat. Its performance was evaluated against incurred-residue swine tissues. This method is based on the cleavage of the disulfide and/or thioester bonds between the metabolites and their conjugate sulfur containing moiety using dithioerythritol to yield desfuroylceftiofur, and further stabilization to desfuroylceftiofur acetamide. The limit of quantitation was 0.1 micrograms ceftiofur equivalents/g tissue. The assay is specific for ceftiofur-related metabolites when evaluated against commercially available antibiotics for swine.

Concentration of ceftiofur metabolites in the plasma and lungs of horses following intramuscular treatment.

Ceftiofur sodium, a broad spectrum cephalosporin antibiotic approved for veterinary use, is metabolized to desfuroylceftiofur which is conjugated to micro as well as macromolecules. Twelve horses, weighting 442-618 kg, were injected intramuscularly with a single dose of 2.2 mg ceftiofur/kg (1.0 mg/lb) body weight. Blood was collected at various intervals over 24 h after treatment. Three groups of four horses each were euthanized and lungs were collected at 1, 12, and 24 h after treatment. The concentration of desfuroylceftiofur and desfuroylceftiofur conjugates in the plasma and lungs was determined by converting them to desfuroylceftiofur acetamide (DCA) and measured DCA by high performance liquid chromatography with UV detection. The average maximum concentration (Cmax) of desfuroylceftiofur and related metabolites in plasma expressed as ceftiofur equivalents was 4.46 +/- 0.93 micrograms/ml occurred at 1.25 +/- 0.46 h after treatment. These concentrations declined to 0.99 +/- 0.16, 0.47 +/- 0.15 and 0.17 +/- 0.02 microgram/ml at 8, 12, and 24 h, respectively. The mean residence time of ceftiofur metabolites was 6.10 +/- 1.27 h. Concentrations of desfuroylceftiofur and desfuroylceftiofur conjugates in the lungs of horses expressed as ceftiofur equivalents were 1.40 +/- 0.36, 0.27 +/- 0.07, and 0.15 +/- 0.08 micrograms/ml at 1, 12, and 24 h, respectively. These concentrations of the drug at 12 and 24 h in lung homogenate were similar but slightly lower than plasma concentrations in the same horses, and the plasma pharmacokinetic values including half-life were similar to those observed at the approved dose of 1.1-2.2 mg ceftiofur/kg body weight administered intramuscularly once daily for 3-5 days in cattle.

Depletion of intramuscularly injected ceftiofur from the milk of dairy cattle.

Ceftiofur sodium, a new broad-spectrum cephalosporin, has been approved in the US, Canada, and several other countries throughout the world to treat bovine respiratory disease in cattle and dairy cows. In Experiment 1, 6 lactating cows were intramuscularly treated with 2.29 mg of [14C]ceftiofur/kg of BW daily for 5 d. In Experiment 2, 30 additional cows at three locations were similarly treated with 2.2 mg of ceftiofur (unlabeled)/kg of BW. Milk was collected every 12 and 24 h after each dose and every 12 h up to 5 d after the last dose. The majority of milk samples, both during treatment (12 and 24 h after each dose) and after the last dose (up to 5 d following ceftiofur treatment), were negative by screening procedures based on microbial inhibition (Delvotest-P, Bacillus stearothermophilus disk assay, and cylinder plate assays). The receptor-binding Charm Test II assay, which has a limit of detection of .005 ppm of ceftiofur, gave positive tests for milk samples up to 48 h following treatment. When the Charm Test II assay is used with .008 IU/ml of penicillin as a positive control, 44% of the samples from individual cows were negative at 12 h posttreatment. Ninety percent of the samples from individual cows were negative at 24 h after the last treatment. The use of ceftiofur in dairy cattle in accordance with the label directions does not result in total residues in milk higher than the FDA-calculated safe concentration of 1-ppm ceftiofur equivalents. The milk from individual cows did not test positive by the commercial screening assays examined in this study, except for the Charm Test II. The Charm Test II was 90% negative using the Charm Sciences criteria at 24 h after the last treatment.