Lincosamides analysis

For the analysis of macrolide and lincosamide residues in liquid foods such as milk, a pretreatment step for fat removal carried out by centrifugation (133-135) is usually required. Semisolid food samples such as muscle, kidney, and liver require often more intensive sample pretreatment including a mincing and/or a homogenization step for breaking up tissue. 

For efficient extraction of macrolide and lincosamide residues from edible animal products, bound residues should be rendered soluble, most if not all of the proteins should be removed, and high recoveries for all analytes should be provided. Since tliese antibiotics do not strongly bind to proteins, many effective extraction methods have been reported. Sample extraction/deproteinization is usually accomplished by vortexing liquid samples or homogenizing semisolid samples with acetonitrile (136—139), acidified (136,140-142) orbasified acetonitrile (143), methanol (14, 144, 145), acidified (145-147) or basified methanol (148), chloroform (149-151), or dichloromethane under alkaline conditions (152). However, for extraction of sedecamycin, a neutral macrolide antibiotic, from swine tissues, use of ethyl acetate at acidic conditions has been suggested (153), while for lincomycin analysis in fish tissues, acidic buffer extraction followed by sodium tungstate deproteinization has been proposed (154). 

In liquid chromatographic analysis of macrolides and lincosamides, most popular is the ultraviolet detector (Table 29.4). Tylosin, tilmicosin, spiramycin, sedecamycin, and josamycin exhibit relatively strong ultraviolet absorption, but erythromycin, lincomycin, pirlimycin, and oleandomycin show extremely weak absorption in the ultraviolet region. Hence, detection at 200-210 nm has been reported for the determination of lincomycin (146). However, a combination of poor sensitivity and interference from coextractives necessitated extensive cleanup and concentration of the extract. Precolumn derivatization of pirlimycin with 9-fluorenylmethyl chloroformate has also been described to impart a chromophore for ultraviolet detection at 264 nm (140). 

Electrochemical detection is better suited to the analysis of erythromycin and lincomycin. This method of detection has been applied for the determination of erythromycin A (139) and lincomycin (154) residues in salmon tissues. Liquid chromatography coupled with mass spectrometry is particularly suitable for confirmatory analysis of the nonvolatile macrolides and lincosamides. Typical applications of this technique are through thermospray mass spectrometry, which has been used to monitor pirlimycin in bovine milk and liver (141,142), and chemical ionization, which has been applied for identification of tilmicosin (151) in bovine muscle, and for identification of spiramycin, tylosin, tilmicosin, erythromycin, and josamycin residues in the same tissue (150). 

Currently, one structure of a Irncosamide antibiotic bound to the ribosome is available for analysis [4]. like the macrolide antibiotics, drndamycin binds near the hydrophobic crevice at the entrance to the peptide exit turmel. As with the macrolide carbomycin A, dindamycin interacts not only with the hydrophobic crevice at the entrance to the peptide exit turmd, but also with the active site hydrophobic crevice. The nudeotides that surroimd the clindamydn binding site were previously implicated in binding of lincosamides based on nucleotide protection studies and on the analysis of mutations conferred by resistance (Fig. 4.4). 

Berryman, D. I., and Rood, J. I. (1989). Cloning and hybridization analysis of ermP, a macrolide-lincosamide-streptogramin B resistance determinant from Clostridium perfringens. Antimicrob. Agents Chemother. 33, 1346-1353. 

Monod, M., Mohan, S., and Dubnau, D. (1987). Cloning and analysis of ermG, a new macrolide-lincosamide-streptogramin B resistance element from Bacillus sphaericus. J. Bacterial. 169, 340-350. 

In 2008, Carretero et al. described a multi-class method for the analysis of 31 antibacterials (including f)-lactams, macrolides, lincosamides, quinolones, sulfonamides, tetracyclines, nitroimidazoles, and trimethoprim) in meat samples by PLE-LC-MS/MS. Meat samples were homogenized and blended with EDTA-washed sand, then extracted with water by applying 1500 psi (Ib/in. ), at 70°C. One extraction cycle was 10 min. A drawback of the method is the large volumes of extracts (40 ml) obtained, which required evaporation to concentrate the extract volume prior to final analysis. This evaporation step considerably increases the time required for sample preparation. The proposed method has been applied to the analysis of 152 samples of cattle and pig tissues, with the presence of quinolones, tetracyclines, and sulfonamides detected in 15% of the samples, although at concentrations below the MRLs. 

As a result of these observations, recommendations have been made to include an incubation step in methods of analysis for lincosamides in liver. " If the aim is to detect lincosamide use, then inclusion of an incubation step is necessary to maximize the possibility of detection. However, as indicated earlier, MRLs, where set, are based on the parent compound only with no reference to the metabolites, either by summation of separate measurements or by conversion to a suitable marker. Thus, inclusion of an incubation step could lead to falsely high residue concentrations and the possibility of samples that fall within the legal limits being reported as non-compliant. As a result, analytically, steps should be taken to minimize enzymatic activity to prevent this reverse metabolism. A long-term solution to this issue might be to include the sulfoxides in the legislation as additional markers in some form. 

Lincomycin (and to some extent the other lincosamides) is commonly mislabeled as a macrolide and as such is often included in multi-residue macrolide procedures. Honey has been the most common target matrix, because of interest in the use of lincomycin for control of foulbrood disease in bees. Extraction from honey has principally been by dissolution/dilution with aqueous solvents. Acetonitrile has been used as extractant for tissues and milk. Dispersion onto sand prior to hot-water extraction has also been used for the analysis of milk. Solid-phase " and liquid-liquid extraction have been used for clean-up. Separation is customarily achieved on reversed phase (C18) columns prior to determination by MS or MS/MS with APCI or more commonly ESI. ... 

---