Chloramphenicol cleanup

Selectivity in lAC depends on the specificity of the immobilized antibody and, thus, monoclonal antibodies are preferentially used. In that case, a large amount of sample can be subjected to immunoaffinity cleanup without any retention of matrix components. This opens the possibility to determine very low concentrations of drug residues in edible animal products. For example, 20 ng chloramphenicol in 1 L milk can be determined with a recovery of 99% when 1 L of defatted milk is submitted to immunoaffinity cleanup. The chromatograms obtained after LC analysis were as clean as those obtained when 10 ml milk containing the same amount of chloramphenicol was also submitted to immunoaffinity cleanup (170). 

Immunoaffinity cleanup was first applied in drug residue analysis for the determination of chloramphenicol in swine muscle tissue by LC (113). The lAC column was prepared using monoclonal antibodies originally developed for an enzyme-linked immunosorbent assay (ELISA) method (171) specific for chloramphenicol. Meat samples were extracted with water, and a concentrated phosphate buffer was added to the filtered extracts before immunoaffinity cleanup. A phosphate buffer was used in the washing process, whereas chloramphenicol was eluted from the column with a glycine/sodium chloride solution of pH 2.8. For subsequent LC analysis, this eluate was extracted with ethyl acetate, evaporated, and reconstimted in the mobile phase. The same analytical scheme was later successfully applied for the determination of chloramphenicol in eggs and milk as well (170, 172). 

Of more immediate interest are approaches that permit offline TLC-MS in which the spots are scraped out from the layer and the analytes are either extracted from the sorbent to be transferred to the mass spectrometer as discrete samples or are introduced without sorbent removal into the spectrometer on a direct insertion probe (51). TLC-MS quantification and confirmation efficiency can be further enhanced by submitting the TLC extract to an additional chromatographic separation using a different technique prior to the final MS analysis. Advantages of this approach over direct TLC-MS include extra cleanup through the additional chromatographic separation (52). This has been realized in the TLC-GC-MS analysis of eggs and meat for chloramphenicol residues (49). 

All radioimmunoassays published thereafter, except those described by Hock and Liemann (38), Freebairn and Crosby (39), and Pohlschmidt et al. (40), were based on a similar procedure (Table 28.2). However, Hock and Liemann (38) applied a more simplified extraction/cleanup procedure for the analysis of chloramphenicol residues in animal tissues, milk, urine, and plasma. In this assay, competitive inhibition between chloramphenicol labeled with " C and antibody has been demonstrated. 

To remove lipids, sample extracts are frequently also partitioned with n-hexane (25, 33-35, 37, 40, 43, 45, 47, 49, 50, 53-57, 62), petroleum ether (31, 38, 63), isooctane (36, 41, 48), or toluene (26, 58, 59, 61). Use of n-toluene is not recommended, however, in chloramphenicol and florfenicol analysis, because these drugs have the tendency to transfer into toluene to some extent during the partitioning process. As an alternative to the classic liquid-liquid partitioning cleanup, some workers in the field (24, 26, 34, 58, 59) have suggested use of diatomaceous earth columns as another option of a liquid-liquid partitioning process that offers substantial reduction in emulsification problems and, thus, allows a high recovery increase. 

Liquid chromatography cleanup on a LiChrosorb Diol column has been further proposed for the offline purification of chloramphenicol residues from bovine muscle and eggs (32). An online approach based on reversed-phase principles has also been described for isolation of chloramphenicol residues from swine kidney by an automated column switching system (63). Use of a protein exclusion column (Hisep) has been also suggested in an online trace-enrichment method for the determination of chloramphenicol in animal tissues (52). By employing a column-switching system, all chloramphenicol that eluted from the protein exclusion column was trapped at the entry of a 5 m Supelcosil LC-18 preconcentration column, to be subsequently back-flashed into the analytical column. 

A specific cleanup procedure based on immunoaffinity chromatography with polyclonal antibodies has been described by Gude et al. (50) for the gas chromatographic determination of chloramphenicol in swine tissues. In this method, tissue sample is extracted with acetonitrile/4% sodium chloride (1 1) Following centrifugation, the supernatant is purified with n-hexane, and chloram-... 

Van de Water, C. Tebbal, D. Haagsma, N. Monoclonal antibody-mediated cleanup procedure for the high-performance liquid chromatographic analysis of chloramphenicol in milk and eggs. J. Chromatogr., 478 205-15. 1989. 

Figure 1 Schematic representation of immunoaffinity column cleanup. CAP, chloramphenicol (an antimicrobial). (Reproduced with permission from Haagsma N (1990) Sample pretreatment in drug residue analysis. In Haagsma N, Ruiter A, and Czedik-Eysenberg PB (eds.) Residues of Veterinary Drugs in Food, Proceedings of the EuroResidue Conference, Noordwijkerhout, The Netherlands, 21-23 May 1990, p. 47. Utrecht Rijksuniver-siteit.)...

Antibiotics The AOAC has listed methods for sulfamethazine residues in swine tissues with determination either by GC-MS or GC-ECD of methylated derivatives and for sulfamethazine (and for the class of sulfonamides) in milk with determination by HPLC-UV. There is an AOAC method for the class of sulfonamide antimicrobials in animal tissues using solvent extraction and liquid partitioning with determination by TLC and fluorimetric scanning. For analysis of tetracyclines, AOAC describes methods based on buffer extraction from tissue samples and SPE (Cis) cleanup, or metal chelate affinity binding from milk samples, with determination in both cases by HPLC-UV. USDA/FSIS methods include (1) a method (similar to the AOAC GC-MS method for sulfamethazine) for confirmation of sulfonamide residues in edible tissues using solvent extraction and multiple liquid partitioning with determination of the methylated derivatives by GC-MS (2) methods for determination and confirmation of chloramphenicol in muscle by solvent extraction, liquid partitioning, and determination of the trimethylsilane (TMS) derivative by GC-ECD and GC-MS, respectively and (3) a method for determination of the beta-lactam antibiotic amoxicillin by aqueous extraction, cleanup by tricarboxylic acid precipitation, and ether extraction and formation of a fluorescent derivative for determination by LC. 

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