Chloramphenicol methods

Briefly stated, the production of chloramphenicol by the surface culture method involves inoculating a shallow layer, usually less than about 2 cm, of a sterile, aqueous nutrient medium with Streptomyces ver)ezuelae and incubating the mixture under aerobic conditions at a temperature between about 20° and 40°C, preferably at room temperature (about 25°C), for a period of about 10 to 15 days. The mycelium is then removed from the liquid and the culture liquid is then treated by methods described for Isolating therefrom tne desired chloramphenicol. 

As in the case of chloramphenicol palmitate, it may be desirable to have the more soluble (less stable) polymorph of a drug. One method, based on thermo-... 

A high performance capillary electrophoresis (HPCE) was described for the separation and simultaneous determination of OTC, TC, CTC, DC, and chloramphenicol in honey. The use of buffer pH 3.2 containing 0.02 mol/L Na2HP04 and 0.01 mol/L citric acid with addition of 4% (v/v) A-methylmorpholine and 12% (v/v) acetonitrile demonstrated a good separation of these five antibiotics within 20 min. The proposed method gave detection limit (signal to noise ratio > 5) of 20 pg/L for OTC [26],... 

It is interesting to note that this method was used in an early analysis of the antibiotic compound chloramphenicol, whose structure includes a nitroaromatic group [30]. 

Other antibiotics studied using RPC include chloramphenicol which could be determined at serum concentrations of about 0.5 /xg/ml using 0.1 ml samples ( 15) after extraction with ethyl acetate (316). A simple method for the analysis of chloramphenicol in serum and cerebrospinal fluid has been reported in which the analyte is extracted into methanol and the extract chromatographed with acetic acid-water methanol (1 62 37) as the mobile phase (i/7). 

Fig. 1. Outline of the strategy to construct GST-fused expression plasmids by the in vitro recombination-assisted method. Am, Gm, and Cm are abbreviations for ampicillin-, gentamicin-, and chloramphenicol-resistance, respectively. The figure also indicates the ccdB gene encoding a toxin targeting the co//essential DNA gyrase and the phage X recombination sites (attB, attP, attL, and attR).

Chemical synthesis of racemates and subsequent resolution via crystallization of diastereomeric salts is employed in the preparation of rf-biotin and tocopherol (vitamins), dexchlorpheniramine (antihistaminic), levomepromazine (neuroleptic), levorphanol (analgesic), and naproxen (antiphlogistic), to note some examples4, threo-2-Amino-1 -(4-nitro-phenyl)-l,3-propanediol, an intermediate in the production of chloramphenicol, is resolved by crystallization with entrainment or via crystallization of the salt with camphorsulfonic acid4. Enzymatic resolutions are increasingly employed, normally via deacetylation of racemic acetates. This method has recently been used in the synthesis of carbacyclin derivatives5. 

Several investigations on the stability of chloramphenicol during thermal treatment have been also carried out. Chloramphenicol has been found quite stable to heating when added to water or milk. Even after 2 hours of boiling, no more than 8% loss of its amount could be observed by physicochemical methods of analysis (16). Application of microbiological methods has shown, however, a 33.3-35% reduction in the activity of chloramphenicol after heating the milk or water samples at 100 C for 30 min (3). 

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). 

If tlie identity of the detected drug residue is still unknown after these tests have been performed, a TLC-bioautography procedure is then applied to isolate and tentatively identify the residues (64, 72). This method is not quantitative and only gives direction to the analyst as to what determinative/confirmatory method of analysis should be used. Following this tentative identification, presumptive positives are then quantified and confirmed by validated physicochemical methods. A TLC method (73) is applied for analyzing presumptive sulfonamide residues, a GC-ECD method (74) followed by a GC-MS NCI method (75) for analyzing chloramphenicol, and LC/UV methods (76,77) for analyzing -lactams and tetracyclines. 

Despite the fact that the preparation of chloramphenicol-specific antibodies was reported as early as in 1966 (36), it was 1984 before the first immunoassay was published for the determination of chloramphenicol residues in swine muscle, eggs, and milk (37). This first-published method was a radioimmunoassay that required an extraction procedure and special laboratory facilities to attain a quantification limit of 1 ppb. Employed polyclonal antibodies showed insignificant crossreactivity with structurally related compounds, except that thiamphenicol that did not interfere with the analysis. However, cross-reactivity was significant for metabolites deviating from the parent compound in the acyl side chain. 

Apart from radioimmunoassays, various enzyme-linked immunosorbent assays have been described as well. Campbell et al. (42) first reported a sensitive and specific ELISA using polystyrene tubes and a polyclonal antibody. However, the performance of this method was not evaluated with real samples but only with standards and aqueous muscle tissue extracts. Sensitive ELISAs were also developed for the determination of chloramphenicol in milk (43) and eggs (44) the results drawn by the latter assay correlated well with those obtained by application of a radioimmunoassay. 

In general, most of published immunochemical methods on chloramphenicol assay are based on antibodies particularly specific for the aromatic ring and the propanediol moiety of chloramphenicol. This is due to the fact that the main chloramphenicol analogues such as thiamphenicol differ from chloramphenicol in one of these parts of the chloramphenicol molecule. To obtain such antibodies, chloramphenicol was linked to the carrier bovine serum albumin at the acyl chain of the molecule by mixed anhydride or carbodiimide reactions (49, 50). In both... 

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. 

---