Ceftiofur analysis

Higher specificity and selectivity can be obtained by reacting -lactams with suitable reagents to form derivatives with improved ultraviolet chromophores. Thus, precolumn derivatization of penicillins with triazole-mercuric chloride and ultraviolet detection at 325 nm (71, 90, 112, 114, 115, 121, 122), 340 nm (123), or 345 nm (116) has become the method of choice for more selective detection and matrix interferences reduction. An alternative precolumn reaction using iodoacetamide as derivatizing reagent has been described in ceftiofur analysis (124), while imidazole-mercuric chloride has also been suggested for on-line postcolumn derivatization of penicillin G (69). 

It has been recently reported (109) that use of both Penase and lactamase II hydrolysis and screening assays prior to chromatographic analysis can tentatively classify -lactams into three subgroups the first group includes a ceftiofur metabolite represented by desfuroyl-ceftiofur-cysteine the second, cephapirin and the third, penicillin G, ampicillin, amoxicillin, and cloxacillin. In this approach, portions of aqueous extracts of tissues are treated separately with Penase and lactamase II, and results are compared with those of untreated samples and positive controls. Bioactive ceftiofur metabolites are present, provided that the extracts retain inhibitory activity after Penase treatment but lose activity after lactamase II treatment and are positive in response to the immunochemical Lac-Tek-Cef test but negative to the Lac-Tek-Bl test (113). This approach can eliminate a large number of negative samples and, therefore, increases the efficiency of the assay. 

Tables 2.20 and 2.21 present estimated data for sales of AMDs for treatment and prevention of BRD in three countries and the EU, together with an analysis of major products used in the United States. The market is dominated by the United States and EU, which together account for 63% of the global BRD market. The US market is dominated by two products containing macrolides (53% of total), three products containing ceftiofur (22.5% of total), and two products containing fluoroquinolones (21.5% of total). While florfenicol has a much smaller percentage share, it is nevertheless signiflcant in view of the market...

Oliveira and Cass described a method for the analysis of cephalosporin antibiotics in bovine milk using RAM columns for on-line sample clean-up. The system was composed of a RAM bovine serum albumin (BSA) phenyl column coupled to a C18 analytical column. Milk samples were directly injected after addition of 0.8 mM solution of tetrabutylammonium phosphate. The standard curve was linear over the range 0.100-2.50 tig/ml for five cephalosporin antibiotics (cefoperazone, cephacetril, cephalexin, cephapirin, and ceftiofur). The limits of quantification and detection reported were 0.100 and 0.050 ttg/ml, respectively. The method showed high intermediate precision [coefficient of variation percent (CV%) 2.37-2.63] and recovery (CV% 90.7-94.3) with adequate sensitivity for drug monitoring in bovine milk samples. 

Ceftiofur is based on 7-aminocephalosporanic acid, which is responsible for the biological activity of the compounds. Only a few methods involving the chemical analysis of ceftiofur are reported in literature. This might be caused by the difficulty of analyzing this compound, which is a result of its metabolism and instability combined with the rather complex EU MRL definition. 

Ceftiofur is known to rapidly metabolize after intramuscular administration, resulting in metabolite residues found in milk and tissue. Reported metabolites include desfuroylceftlofur (DEC), desfuroylceftlofur cysteine disulfide (DCCD), protein-bound DEC, and ceftiofur thiolactone. " Because these metabolites are all micro-biologically active, the EU MRL was defined as the sum of all residues retaining the f)-lactam structure, expressed as DEC, " whereas the Codex Alimentarius Commission (CAC) defined DEC as the only marker residue, simplifying the analysis. This approach is also used in the United States of America. 

Three main approaches for the analysis of ceftiofur are reported. The first approach, which is reported for... 

The analysis of ceftiofur by applying a deconjugation using dithioerythritol is reported for plasma, milk, and tissue samples. Dithioerythritol, a reducing agent that causes cleavage of disulfide bonds, is added to the sample extract to release bound ceftiofur and its metabolites from proteins. Next, iodoacetamide is added to derivatize the free ceftiofur and its metabolites to desfuroylceftiofur... 

A clean-up procedure for the analysis of ATMA as a marker compound for ceftiofur and ceftiofur-related metabolites, based on ion exchange SPE, has been developed. Separation was carried out using reversed phase FIPLC and detection by LC-MS/MS. At the time when this chapter went to press (December 2010), this method had not yet been tested using ceftiofur incurred tissue material, and therefore this new approach has not yet been compared to current approaches. 

For ceftiofur, the EU MRL includes ceftiofur and all active ceftiofur metabolites. To be able to comply with this MRL definition, all active metabolites of ceftiofur have to be included in the analysis method. 

Berendsen B, Essers M, Mulder P, et al.. Newly identified degradation products of ceftiofur and cephapirin impact the analytical approach for quantitative analysis of kidney, J. Chromatogr. A 2006 1216 8177-8186. 

Baere SD, Pille P, Croubels S, et al. High-performance liquid chromatographic-UV detection analysis of ceftiofur and its active metabolite desfuroylceftiofur in horse plasma and synovial fluid after regional intravenous perfusion and systemic intravenous injection of ceftiofur sodium. Anal ChimActa. 2004 512 75-84. 

Tyczkowska, K.L. Voyksner, R.D. Straub, R.F. Aronson, A.L. Simultaneous multiresidue analysis of 8-lactam antibiotics in bovine milk by liquid chromatography with ultraviolet detection and confirmation by electrospray mass spectrometry. JAOAC Int, 1994, 77, 1122-1131 [milk cow LC-MS electrospray UV detection LOD 10 ppb extracted ampicillin, ceftiofur, cephapirin, cloxacillin, penicillin G column temp 40]... 

Desacetylcephapirin was extracted from milk samples and quantitated using a PLRP-S column (A = 290 nm) using a 16/84 acetonitrile/water (10 mM H3PO4 with lOmM KH2PO4 and lOmM sodium decanesulfonate) mobile phase [1379], This study also tabulated the conditions for the analysis of amoxicillin, ampicillin, cephapirin, ceftiofur, cloxacillin, and penicillins G and V. Again a C,g and an 30% acetonitrile/water (phosphate buffer) combination were used in each case. 

The major limitation of QqQ working in the MRM mode is that a complete mass spectrum cannot be obtained, so it is difficult to detect the presence of metabolites or nontarget compoimds. This concern is important when metabolites are explicitly included in the list of the regulated substances, (e.g., benzoimidazoles, carprofen, ceftiofur, and florfenicol) [11]. Conversely, the classical tridimensional ion trap analyzer has been used for this purpose [34], because it provides high sensitivity in full-scan mode and has the ability to perform multiple-stage fragmentations (MS" ), but it does not have adequate sensitivity and reliability to perform quantitative trace analysis. 

X. Jiang, H. Yang, B. Dettman and M. P. Doyle, Analysis of fecal microbial flora for antibiotic resistance in ceftiofur-treated calves, Food-borne Pathog. Dis., 2006, 3, 355-365. 

---