Chloramphenicol determination

Procedures for the determination of chloramphenicol concentrations in blood serum include HPLC and immunoassay. Methods for chloramphenicol determination must be able to differentiate between the prodrug forms, chloramphenicol palmitate or succinate, and their active metabolite, chloramphenicol. (An HPLC method for measuring chloramphenicol is included on this book s accompanying Evolve site, found at http //evoIve. elsevier.com/Tietz/textbook/.)... 

Optical detection of an antibiotic, chloramphenicol, based on competitive displacement of a chloramphenicol-methyl red conjugate bound to a chloramphenicol-imprinted polymer with free chloramphenicol has been demonstrated [40]. A flow injection system in conjunction with a 10 cm stainless steel column packed with the imprinted polymer and acetonitrile as a carrier solution containing chloramphenicol-methyl red conjugate was constructed. The dye conjugate released by displacement by free chloramphenicol was monitored at 460 nm. The signals were proportional to the concentration of free chloramphenicol injected, and the calibration range of this system included the therapeutic range of a chloramphenicol. This concept of flow displacement systems could be applicable not only for chloramphenicol determination but also for other template molecules. 

The efficacy of florfenicol in vivo was determined by measuring the dose required to obtain values for protection from infection in 50% of the animals (PD q) against 10 chloramphenicol-resistant strains and two chloramphenicol-sensitive isolates. Florfenicol, chloramphenicol, and thiamphenicol were evaluated concurrendy against each strain. Against sensitive Enterobacter 50 subcutaneous and oral routes were similar for dorfenicol and... 

Liquid chromatographic determination of chloramphenicol in kidney tissue homogenates using valve-switching techniques , 113 171 -174(1988). 

Antibiotics may be classified by chemical structure. Erythromycin, chloramphenicol, ampicillin, cefpodoxime proxetil, and doxycycline hydrochloride are antibiotics whose primary structures differ from each other (Fig. 19). Figure 20 shows potential oscillation across the octanol membrane in the presence of erythromycin at various concentrations [23]. Due to the low solubility of antibiotics in water, 1% ethanol was added to phase wl in all cases. Antibiotics were noted to shift iiB,sDS lo more positive values. Other potentials were virtually unaffected by the antibiotics. On oscillatory and induction periods, there were antibiotic effects but reproducibility was poor. Detailed study was then made of iiB,sDS- Figure 21 (a)-(d) shows potential oscillation in the presence of chloramphenicol, ampicillin, cefpodoxime proxetil, and doxycycline hydrochloride [21,23]. Fb.sds differed according to the antibiotic in phase wl and shifted to more positive values with concentration. No clear relationship between activity and oscillation mode due to complexity of the antibacterium mechanism could be discovered but at least it was shown possible to recognize or determine antibiotics based on potential oscillation measurement. 

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],... 

Nakagawa, T., Masada, M. and Uno, T.J. (1975) Gas chromatographic determination and gas chromatographic-mass spectrophotometric analysis of chloramphenicol, thiamphenicol and their metabolites. Journal of Chromatography, 111, 355. 

Wang, A. B., Zhang, L., and Fang, Y. Z. (1999). Determination and separation of chloramphenicol and its hydrolysate in eye-drops by capillary zone electrophoresis with amperometric detection. 

Takino, M., Daishima, S., and Nakahara, T. (2003). Determination of chloramphenicol residues in fish meats by liquid chromatography-atmospheric pressure photoionization mass spectrometry.. Chromatogr. A 1011, 67 75. 

The primary application of the procedure is the determination of the presence or absence of 3-lactam 7) residues in milk and secondarily to measure the levels quantitatively. The receptor assay system has now been expanded to qualitatively detect residues of tetracycline, erythromycin, streptomycin, chloramphenicol, novobiocin, and sulfamethazine in milk, serum and urine (Table II) (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). 

Law et al. [15] determined the diffusion coefficient for benzyl penicillin in thin films of Palacos, Simplex and CMW cements assuming that antibiotic transport can be described by Fick s law using a finite difference approximation to quantify transient non-steady-state behaviour. These investigators found that the diffusion coefficient was increased in the presence of additives and proposed that the finite difference approach could be applied to determine release of antibiotic from preloaded PMMA beads. Dittgen and Stahlkopf [16] showed that incorporation of amino acids of varying solubilities also affected release of chloramphenicol from polymethacrylic... 

The ability of liver to biotransform chloramphenicol has been also demonstrated in several fish species. In pertinent studies, various metabolic pathways were determined and chloramphenicol-glucuronide, chloramphenicol-base, chloramphenicol-alcohol, and chloramphenicol-oxamate were the main metabolites observed (34, 35). Following hepatic biotransformation, a large proportion of the administered dose was excreted in the urine. 

Results showed a total of 2.8% of the samples (n 2972) to be inhibitor positive by the Delvotest SP test further examination identified 1.7% as -lactam antibiotics, and 1.1 % as sulfonamides and dapsone. The percentage of chloramphenicol suspicious samples determined by the Charm II test was amazingly high however, tests for confirmation were not available and contamination of the samples by residues of the chloramphenicol-based preservative azidiol could not be excluded with certainty. Low concentrations of streptomycins were also detected in 5.7% of the samples (n 1221), but the MRL was not exceeded. Macrolide and tetracycline residues were not found in significant levels. Model trials with commercially applied yoghurt cultures confirmed how important the compliance to MRLs can be to dairy industry compared to antibiotic-free milk, a pH of 5.0 was reached with a delay of 15 min in the case of contamination with cloxacillin 30 min in the case of penicillin, spiramycin, and tylosin and 45 min in the case of oxytetracycline contamination. 

Apart from the pathophysiological condition of the animal, the mode of drug application may also significantly influence the pharmacokinetic profile of a drug (48, 49). For example, drug residues may persist at the injection site for prolonged periods of time (2). In a study in which various sulfonamides and trimethoprim were injected intramuscularly into swine, detectable residues were found at most sites 6 days after the injection, and with the sulfonamides at 30 days in almost half of the animals (50). Other drugs such as dihydrostreptomycin persist for up to 60 days, while positive residues of chloramphenicol are found at 7 days postinjection. Sodium and procaine penicillin, neomycin, tylosin, and oxytetracycline residues have also been determined at 24 h or more postinjection (51). 

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). 

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