Chloramphenicol confirmation

Florfenicol iahibited 91% of the 399 bacterial isolates at a concentration of 12.5 )J.g/mL (31). At the same concentration, chloramphenicol and thiamphenicol iahibited only 70% and 24% of the isolates, respectively. Other work has also confirmed the superior activity of fiorfenicol against chloramphenicol-resistant strains (32—35). More recendy it has been shown that fiorfenicol is active against E. coli strains that produce type I, II, or III CAT enzymes (36). 

A famous example of the use of nitro compounds in synthesis was the original synthesis of the antibiotic chloramphenicol (8), which is still used to treat tropical diseases. This synthesis also confirmed the structure of chloramphenicol and established that the (-)-thrco compound was the biologically active stereoisomer. 

Typhoid fever caused by Salmonella typhi or S. paratyphi is an important and prevalent cause of continuous fever without localizing symptoms in the tropics. The diagnosis can be confirmed with a bloodculture. Response on therapy is often seen only after 3 days when the fever subsides. Chloramphenicol-resistant Salmonella typhi was first described in Vietnam in 1973. Its prevalence reached 95% in the 1970s and then decreased to 54% in the 1980s after cotrimoxazole became the treatment of choice. In the mid-1993, there was a dramatic increase in the number of strains of S. typhi, isolated in the hospital and from patients in the outbreaks, which are resistant to the three first-line antibiotics chloramphenicol, cotrimoxazol and ampicillin. This indicated that there was an urgent need for effective antibiotics for the treatment of typhoid fever. 

The resulting disease is fatal in approximately 70% of the cases and those who recover experience a high incidence of acute leukemia (31). It has been reported that a 6-year-old girl died after receiving only 2 g chloramphenicol (32), while a 73-year-old woman died following an estimated total dose of only 82 mg chloramphenicol received as an ophthalmic drug (33). These total dosages have the potential to be present in human food it has been confirmed in a report in which chloramphenicol residues were found in 13 of 3020 calves tested (31). Ten muscle samples had levels above 1 ppm, while one was reported to contain about 12 ppm chloramphenicol. 

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. 

In the following years, the survey for drug residues continued but focused on veterinary drugs commonly used in animal husbandry such as sulfonamides and tetracyclines, and on chloramphenicol. Screening was carried out using the Charm 11 test, while confirmation of positive results involved HPLC or GC-MS. The results obtained are summarized in Table 13.27. 

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). 

Quantification and confirmation of tetracycline, oxytetracycline, and chlortetracycline residues in milk (84) as well as chloramphenicol residues in calf muscle (85) have been also carried out using LC-PB-NCI-MS. Use of an SIM mode allowed a detection limit of about 100 ppb for the tetracyclines and 2 ppb for chloramphenicol residues. 

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. 

Since amphenicols exhibit strong ultraviolet absorption, they are ideal for direct determination by liquid chromatography, without any need for derivatization (Fig. 29.2.1). Their detection wavelengths have been set at 224 nm for thiamphenicol, 220 or 225 nm for florfenicol, and 270-290 nm for chloramphenicol (Table 29.2). Use of photodiode array detectors has been suggested for tentative confirmation of the identity of chloramphenicol residues analyzed by liquid chromatography (26, 37, 38, 40, 53, 58, 59, 61, 63, 66, 67). 

B Roudaut. High performance liquid chromatography with UV detection and scanning UV confirmation of chloramphenicol in fatty liver. J Liq Chromatogr 19 1097-1105, 1996. 

Impens, S., W. Reybroeck, J. Vercammen, et al. 2003. Screening and confirmation of chloramphenicol in shrimp tissue using ELISA in combination with GC-MS2 and LC-MS2. Anal. Chim. Acta 483 153-163. 

Inhibitions of protein synthesis were subsequently confirmed by chemical analyses for protein in growing bacterial cultures38, 39. Figure 439 shows the specific inhibition of protein biosynthesis by chloramphenicol in E. coli and the continuation of RNA ( ribose ) and DNA biosynthesis for an experimental period of 50 minutes. Similar results have been typically obtained with other inhibitors of protein biosynthesis. 

Salmonella typhimurium DT104 is usually resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. An outbreak of 25 culture-confirmed cases of multidrug-resistant S. typhimurium DT104 has been identified in Denmark (54). The strain was resistant to the above-mentioned antibiotics and nalidixic acid and had reduced susceptibility to fluoroquinolones. A swineherd was identified as the primary source (54). The DT104 strain was also found in cases of salmonellosis in Washington State, and soft cheese made with unpasteurized milk was identified as an important vehicle of its transmission (55). 

Amichai B, Grunwald MH, Halevy S. Erythema multiforme resulting from chloramphenicol in eye drops confirmation by mast cell degranulation test. Ann Ophthalmol Glaucoma 1998 30 225-7. 

Liquid chromatography-thermospray-mass spectrometry has been successfully applied for the detection/ confirmation of nicarbazin residues in chicken tissues using negative-ion detection in SIM mode. Liquid chromatography-thermospray-mass spectrometry in SIM mode has also been used for the quantification of residues of moxidectin in cattle tissues and fat, ° and nitroxynil, rafoxanide, and levamisole in muscle. Confirmatory methods based on LC-TSP-MS have been further reported for the determination of penicillin G, cephapirin, and various penicillin derivatives " in mUk. Comparative evaluation of the confirmatory efficiency of LC-TSP-MS and LC-TSP-MS-MS in the assay of maduramycin in chicken fat showed the former approach to be marginally appropriate, whereas the latter is highly efficient. Tandem LC-MS-MS has also been successfully applied in the analysis of residues of chloramphenicol in milk and fish. ... 

The pathways of polymorphic transitions for cephalexin, chloramphenicol palmitate, and indomethacin have been studied, and the composition of partially transformed samples determined by XRPD.27 The metastable forms of chloramphenicol palmitate were found to transform into the stable phase when found at room temperature, confirming the results of a previous study.28 In contrast, cephalexin became amorphous upon grinding. Indomethacin exhibited the interesting behavior of becoming amorphous when ground at 4°C, but could be transformed into a metastable phase when ground at 30°C. 

Neither doxycycline nor a fluoroquinolone may reach therapeutic levels in the cerebrospinal fluid. Therefore, ciprofloxacin augmented with chloramphenicol, rifampin, or penicillin is the treatment of choice for suspected or confirmed anthrax meningitis (4). 

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