A determinative and confirmatory method for ceftiofur metabolite desfuroylceftiofur cysteine disulfide in bovine kidney by LC-MS/MS.

Ceftiofur is a cephalosporin ß-lactam antibiotic widely used for treating certain bacterial infections in beef and dairy cattle. The regulatory HPLC-UV method for ceftiofur residues in animal tissues is time consuming and non-specific. Additionally, because the regulatory method involves chemical reactions to convert the metabolites into a single moiety, it is virtually impossible to incorporate the procedure into a multi-residue method. Ceftiofur residue violations in beef and dairy cattle have been frequently reported and therefore an improved method is needed. Herein we report a rapid and sensitive LC-MS/MS method for the determination and confirmation of ceftiofur metabolite, desfuroylceftiofur cysteine disulfide (DCCD), in bovine kidney tissue. The new method utilizes a simple extraction with phosphate buffer followed by SPE cleanup. A deuterated internal standard was synthesized and used for quantitation. The matrix-based calibration curve was linear from 25 to 2000 ng/g. The average accuracy for control kidney samples from six different sources fortified at 50-1000 ng/g was 97.7-100.2% with CV ≤ 10.1%. The limit of confirmation was 50 ng/g.

A No Observable Adverse Effects Level (NOAEL) for pigs fed melamine and cyanuric acid.

Ingesting melamine adulterated milk products led to kidney stones in many infants in 2008. This differs from the renal failure caused by intratubular crystal formation after co-ingestion of melamine (MEL) and cyanuric acid (CYA) in adulterated pet foods in 2007. To better understand the potential risk of developing crystal nephropathy following co-ingestion of MEL and CYA, we fed 16 weanling pigs 0, 1, 3.3, 10, 33, or 100 mg/kg bw/day of each MEL and CYA, or 200 mg/kg bw/day of either compound individually for 7 days. Crystals were found in the renal medulla and cortex and urine sediments of all pigs fed both MEL and CYA each at 10 mg/kg bw/day (or greater). Crystals were also found in one of the two pigs fed 200 mg/kg bw/day MEL-only. In a 28 day study, 36 weanling pigs were fed 0, 1, or 3.3 mg/kg bw/day of MEL and CYA or 200 mg/kg bw/day MEL-only. Only one of the 3.3 mg/kg MEL and CYA pig kidneys contained crystals. The no-observed-adverse-effect level (NOAEL) for pigs fed MEL and CYA for 28 days was concluded to be 1.0 mg/kg bw/day corresponding to 25 mg/kg (ppm) MEL and 25 mg/kg (ppm) CYA in dry feed.

The proteomic advantage: label-free quantification of proteins expressed in bovine milk during experimentally induced coliform mastitis.

Coliform mastitis remains a primary focus of dairy cattle disease research due in part to the lack of efficacious treatment options for the deleterious side effects of exposure to LPS, including profound intra-mammary inflammation. To facilitate new veterinary drug approvals, reliable biomarkers are needed to evaluate the efficacy of adjunctive therapies for the treatment of inflammation associated with coliform mastitis. Most attempts to characterize the host response to LPS, however, have been accomplished using ELISAs. Because a relatively limited number of bovine-specific antibodies are commercially available, reliance on antibodies can be very limiting for biomarker discovery. Conversely, proteomic approaches boast the capability to analyze an unlimited number of protein targets in a single experiment, independent of antibody availability. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), a widely used proteomic strategy for the identification of proteins in complex mixtures, has gained popularity as a means to characterize proteins in various bovine milk fractions, both under normal physiological conditions as well as during clinical mastitis. The biological complexity of bovine milk has, however, precluded the complete annotation of the bovine milk proteome. Conventional approaches to reducing sample complexity, including fractionation and the removal of high abundance proteins, has improved proteome coverage, but the dynamic range of proteins present, and abundance of a relatively small number of proteins, continues to hinder comparative proteomic analyses of bovine milk. Nonetheless, advances in both liquid chromatography and mass spectrometry instrumentation, including nano-flow liquid chromatography (nano-LC), nano-spray ionization, and faster scanning speeds and ionization efficiency of mass spectrometers, have improved analyses of complex samples. In the current paper, we review the proteomic approaches used to conduct comparative analyses of milk from healthy cows and cows with clinical mastitis, as well as proteins related to the host response that have been identified in mastitic milk. Additionally, we present data that suggests the potential utility of LC-MS/MS label-free quantification as an alternative to costly labeling strategies for the relative quantification of individual proteins in complex mixtures. Temporal expression patterns generated using spectral counts, an LC-MS/MS label-free quantification strategy, corresponded well with ELISA data for acute phase proteins with commercially available antibodies. Combined, the capability to identify low abundance proteins, and the potential to generate temporal expression profiles, indicate the advantages of using proteomics as a screening tool in biomarker discovery analyses to assess biologically relevant proteins modulated during disease, including previously uncharacterized targets.