Macrolides, detection

However, recent investigations on the effect of the tissue matrix on the detection limits attained by this test have indicated that ceftiofur, sulfonamides, streptomycin, and some macrolide antibiotics cannot be detected in intact meat with the plates and the bacterial strains prescribed in the European four-plate test (81, 82). Two plates of this system were not found suitable for screening sulfamethazine or streptomycin at levels far above the MRL the third plate detected tetracyclines and -lactams up to the MRL levels whereas the fourth was sensitive to -lactams and some but not all macrolides. Detection, on the other hand, of the fluoroquinolones enrofloxacin and ciprofloxacin could only be made possible by an additional Escherichia coli plate not included in the four-plate test. 

Hydrolysis of macrolides by products of the ere genes detected in enterobacteria is only of scientific interest, while esterases VGB-A and VGB-B encoded by the vgb type genes mediate clinically relevant resistance in staphylococci to the B compound (quinupristin) of the streptogramin combination quinu-pristin-dalfopristin. 

Macrolide antibiotics (clarithromycin, dehydroerythromycin, etc.) and sulfonamides (sulfamethoxazole, sulfadimethoxine, sulfamethazine, and sulfathi-azole) are the most prevalent antibiotics found in the environment with levels around a few micrograms per liter, whereas fluoroquinolones, tetracyclines, and penicillins have been detected in fewer cases and usually at low concentrations (nanograms per liter) [3,20,23,72]. This result is not surprising, since penicillins are easily hydrolyzed and tetracyclines readily precipitate with cations such as calcium and are accumulated in sewage sludge or sediments. Several reviews have reported the environmental occurrence of different antibiotics in aquatic and soil compartments. Some of these data are detailed in Table 1. 

Regarding sensors, Draisci et al. [100] reported the development of an electrochemical competitive ELISA for the detection of erythromycin and tylosin in bovine muscle. They used MAbs against these two macrolides and the activity... 

The Charm II 6600/7600 system is the only commercial immunoassay test available for the detection of several macrolides in different matrices (see Table 4). The LOD of erythromycin in milk is 40 pg L 1 and 100 pg L 1 in tissues. 

Erythromycin, a macrolide antibiotic, lacks a significant chromophore. Detection sensitivity was enhanced by using a wavelength of 200 nm and selecting an injection solvent of lower conductivity than the BGE. In order to facilitate the separation of erythromycin and its related substances, 35% (v/v) ethanol was incorporated into a 150 mM phosphate buffer pH 7.5. Resolution of all of the compounds was achieved in approximately 45 min. The method was employed as an assay method for erythromycin and for impurity determination. Peptide antibiotics, such as colistin and polymyxin, are mixtures of many closely related compounds. A validated CZE method for impurity analysis of polymyxin B was described, employing 130 mM triethanolamine-phosphate buffer at pH 2.5 to reduce the adsorption of analyte onto the capillary wall. Methyl-/l-cyclodextrin (M-/1-CD) and 2-propanol were found to be necessary for selectivity enhancement. Using similar buffer additives, the same group developed and validated a method for colistin analysis. ... 

Siemann M, Andersson LI, Mosbach K. Separation and detection of maciolide antibiotics by HPLC using macrolide-imprinted synthetic polymers as stationary phases. J Antibiot 1997 50 89-91. 

Other sources of PPCP contamination to groundwater can originate from farms, leaking septic tanks, and lagoons. For instance, Campagnolo et al. (2002) detected several types of antibiotics including macrolides, tetracycline, sulfonamides, and (3-lactams in groundwater samples collected from sites that were in proximity of a swine farm. 

Maximal plasma concentrations occur 2 to 3 hours after oral administration of reboxetine (178). Reboxetine has linear pharmacokinetics over its clinically relevant dosing range and a half-life of approximately 12 hours. For this latter reason, a twice a day, equally divided dosing schedule was used during clinical trial development. Its clearance is reduced and half-life becomes longer as a function of advanced age (mean = 81 years of age) and renal and hepatic impairment ( 178, 322, 323). Reboxetine is principally metabolized by CYP 3A3/4 such that its dose should be reduced when used in combination with drugs that are substantial inhibitors of CYP (e.g., certain azole antifungals, certain macrolide antibiotics). Reboxetine itself, however, does not cause detectable inhibition of CYP 3A3/4 based on formal in vivo pharmacokinetic interaction studies as well as its own linear pharmacokinetics. 

Taniguchi, K. Nakamura, A. Tsurubuchi, K. O Hara, K. Sawai, T. Identification of Escherichia coli clinical isolates producing macrolide 2 -phos-photransferase by a highly sensitive detection method. FEMS Microbiol. Lett., 167, 191-195 (1998)... 

In general, the macrolides are administered orally but sometimes also paren-terally. All the members of this group are well absorbed and are distributed extensively in tissues, especially in the lungs, liver, and kidneys, with high tissue to plasma ratios. They are retained in the tissues for long periods after the levels in the blood have ceased to be detectable. Elimination of all macrolides occurs primarily through hepatic metabolism, which accounts for approximately 60% of an administered intravenous dose the remainder is excreted in active form in the urine and bile. With oral and intramuscular administration, urinary excretion decreases, but biliary excretion and hepatic metabolism increase proportionally. Milk has often macrolide concentrations severalfold greater than in plasma (7). 

Sedecamycin is a macrolide primarily used for treating swine dysentery. As with most macrolides, sedecamycin is extensively metabolized in swine 20 metabolites have been detected, the major ones being lankacidin C, lankacidinol, and lankacidinol A (106). 

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 Australia, the general antimicrobial screen is performed on kidney and is able to detect -lactam, aminoglycoside, tetracycline, and macrolide antimicrobials and to identify the class of antimicrobial compound present. Where the screen test identifies a class of compounds, confirmation and quantitation are done by the specific HPLC or gas chromatographic (GC) method appropriate for the class of antimicrobial. 

Microbial inhibition tests are extremely sensitive for -lactam antibiotics, primarily penicillin, but mostly are more than 100-fold less sensitive for other commonly used antibacterials such as macrolides, sulfonamides, tetracyclines, or chloramphenicol (4, 5). Therefore, inhibition tests usually classify residues as belonging to the -lactam group. Antibiotics other than -lactams and sulfonamides can be detected by use of the enzyme penicillinase and aminobenzoic acid, respectively (1, 6). 

Since 1974, Bacillus subtilis EGA has been officially employed as the test organism in the German Hemmstoff test to detect residues of tetracyclines, -lactams and aminoglycosides in kidney and muscle tissues with high sensitivity (72). Macrolides can be also detected, but to a lesser extent, whereas chloramphenicol and sulfonamides are difficult to detect. For better detection of sulfonamides, a modification of this test, the German three-plate inhibition test, was developed. This test is based on the same test organism but uses three pH values (6, 8, and 7.2), with the addition of trimethoprim. The pH relationship between the three... 

The four-plate test was initially based on the German Hemmstoff-test with an additional plate of Sarcina lulea at pH 8.0, designed for the detection of lower levels of macrolides, and a fourth plate of Escherichia coli at pH 7.2 for the detection of sulfonamides (74,75). The modified version adopted by the European Community for screening carcasses is based on three plates with Bacillus subtilis BGA at pH values of 6.0, 8.0, and 7.2 with added trimethoprim, respectively, and a fourth plate with Micrococcus luteus NCTC 8340 at pH 8.0 (74). This test as described elsewhere (76) is intended to detect residues of -lactams, tetracyclines, aminoglycosides, sulfonamides, and macrolides in muscle tissue of slaughtered animals, without any prior extraction or cleanup. 

The NKDT test is sensitive to residues of -lactams, tetracyclines, and macrolides. It is more sensitive to sulfonamides than the German three-plate test or the EU four-plate test, but it is relatively insensitive to aminoglycosides. With respect to MRLs set for liver or kidney, the NKDT is too insensitive for aminoglycosides, sulfonamides, and macrolides. One analyst can complete 150-200 tests per day. Due to the high ratio of residue levels in preurine and muscle, a negative result of this test implies that residue levels in muscle are below the limit of detection. 

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). 

The HPLC-receptorgram assay combined the advantages of HPLC separation with the multiresidue detection of the Charm II tests. The procedure was tested for identification and quantitation of the most common veterinary drugs at regulatory levels or lower. It was validated for 40 individual drugs from seven antibiotic families 10 /3-lactams, 13 sulphonamides, 8 tetracyclines, 4 macrolides, 3 amphenicols, and other miscellaneous antimicrobials. This procedure combined a simple aqueous extraction and SPE with HPLC fractionation of individual drugs. Final identification and quantitation was achieved with the Charm II test. A drug contaminant could be identified in less then 3 hours (50). 

Gonzalez de la Huebra, M. J., Bordin, G., and Rodriguez, A. R. (2003). Comparative study of coulometric and amperometric detection for the determination of macrolides in human urine using high-performance liquid chromatography. Anal. Bioanal. Chem. 375 1031-1037. 

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