Tylosin methods

Fig- 5-7 Results (lU/mg) of an interlaboratory study to determinate the potency of tylosin CRS 1 by microbiological assay (diffusion method). 

The application of chromatographic methods to analyze the residues of macrolide antibiotics has been very limited. Moats (85) listed four TLC and one HPLC method for tylosin and one TLC method for erythromycin. In a comparison of the HPLC and microbiological methods for incurred residues in swine, the HPLC method was more sensitive and usually gave higher results (86). 

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). 

Vanillin in MeOH and sulphuric acid was used for the derivatization of MON. Chicken tissue (muscle, liver, skin with adhering fat tissues) samples were homogenized with MeOH-water, NaCl was added to the supernatants, and MON was isolated and concentrated by liquid-liquid partition carbon tetrachloride and by SPE on the silica gel. Standard recoveries ranged from 82% to 96%. The method is specific for MON in the presence of closely related PETs— NAR and SAL. Lasalocid and other antibiotics, such as tylosin, nicarbazin, bacitracin, lin-comycin, and bambermycin, do not react in the system and therefore do not interfere (102). A similar method was also used for the determination of MON in bovine tissues and milk. The homogenization of milk was performed by using MeOH. Recoveries achieved were 79-88% with RSD values of 4.6-9.1% (103). 

Chemical acylation of tylosin subsequently yielded a larger series of esters from which 4"-0-(4-methoxyphenylacetyl)tylosin (15) Figure 5.6) has emerged to the stage of preclinical studies due to its activity against certain resistant organisms and its improved metabolic stability [78-80], Several tylosin-related factors have also been acylated by chemical and biochemical methods [81-84], In addition to esters, 4"-0-alkyl and 4"-deoxy derivatives of tylosin have been prepared, but none was superior to compound (15) [85],... 

Dubois et al. [59] determined the macrolides tylosin, tilmicoson, spiramycin, josamycin, and erythromycin in swine and bovine muscle, kidney and liver tissue, in bovine milk, and in hen eggs, using roxithromycin as IS. The method involves extraction in a Tris buffer, protein precipitation, SPE clean-up on a Oasis HLB cartridge, and LC-MS-MS analysis in SRM mode. All analytes were confirmed by four ions with an ion-ratio reproducibility ranging from 2.4 to 15%. The sample throughput is 50 samples per analyst per day. Draisci et al. [60] developed a confirmatory method for tylosin, tilmicosin, and erythromycin in bovine muscle, liver, and kidney. The quantification limits were 30, 20, and 50 pg/kg in mnscle, 40, 150, and 50 pg/kg in liver, and 40, 150, 80 pg/kg in kidney for tylosin, tilmicoson, and eiythromycin, respectively. Horie et al. [61] reported the multiresidne determination of erythromycin, oleandromycin, litasamycin, josamycin, mirosamycin, spiramycin, tilmicoson, and tylosin in meat and fish. The LOQ was 10 pg/kg in positive-ion LC-ESI-MS in SIM mode. 

Fig. 3. Phylogenetic analysis of acyltransferases. Phylogenetic tree of amino acid sequences of acyl-transferase domains from actinomycete type I PKSs, Multiple alignment and phylogenetic analysis using the bootstrapping method were performed by using CLUSTALW. AVE, avermectin PKS module ERY, erythromycin PKS module FKB, FK520 PKS module MEG, megalomicin PKS module NID, nidda-mycin PKS module NYS, nystatin PKS module OEM, oligomycin PKS module PIK, pikromycin PKS module RAP, rapamycin PKS module RIF, rifamycin PKS module TYL, tylosin PKS module.

International Union of Pure and Applied Chemistry (lUPAC) names are based on a method that involves selecting the longest continuous chain of carbon atoms, and then identifying the groups attached to that chain and systematically indicating where they are attached. Continuing with tylosin as an example, the lUPAC name is [ 2R, iR,AE, 6 ,9/ ,ll/ ,125,135,14/ )-12- [3,6-dideoxy-4-0-(2,6-dide oxy-3- C -methyl- a-L-ribohexopyranosyl)-3- (dimethylami no)- 3-D-glucopyranosyl]oxy -2-ethyl-14-hydroxy-5, 9,13-trimethyl- 8,16-dioxo-l 1- (2-oxoethyl)oxacyclohexadeca-4,... 

Ding et al. described an automated on-line SPE-LC-MS/MS method for the determination of macrolide antibiotics, including erythromycin, roxithromycin, tylosin, and tilmicosin in environmental water samples. A Capcell Pak ME Ph-1 packed-column RAM was used as SPE column for the concentration of the analytes and clean-up of the sample. One millilitre of a water sample was injected into the conditioned SPE column, and the matrix was washed out with 3 ml high-purity water. By rotation of the switching valve (see Fig. 4.2), macrolides were eluted in the back-flush mode and transferred to the analytical column. The limits of detection and quantification obtained were 2-6 and 7-20 ng/1, respectively, which is suitable for trace analysis of macrolides. The intra- and inter-day precisions ranged within 2.9-12% and 3.3-8.9%, respectively. At the three fortification concentrations tested (20, 200, and 2000 ng/1), recoveries of macrolides ranged from 86.5% to 98.3%. 

The 3- and/or 4"-0-acyl derivatives of several tylosin-related macrolides have also been prepared by analogous bioconversion methods the 3-0-acetyl-4"-0-isovaleryl derivative of macrocin (3" -0-demethyltylosin) had properties similar to those of the corresponding ester of tylosin [90]. This research has now been further extended with the reported cloning of a gene encoding the 4"-0-isovaleryl acylase enzyme and its expression by formation of a new hybrid macrolide, 4"-0-isovalerylspiramycin [91]. These results indicate that future contributions can be anticipated from the area of molecular genetics on biochemical processes involved in macrolide biosynthesis, with the promise of production of novel hybrid macrolide structures. 

Complementary to the bioconversion studies on tylosin, chemical methods for selective esterification of its 4"-hydroxyl group were developed in order to synthesize additional esters for structure-activity studies which were not available from bioconversion methods [100,101]. Although deacylation of the 4"-0-acyl group has generally been encountered in vivo, increased resistance to such hydrolysis by mouse liver esterase has been recently reported for two 4"-esters of tylosin [101, 102]. 

Epoxidation of the dienone system in tylosin-related macrolides has been shown to yield the 12,13-P-epoxyenone system [103]. Methods for conversion of the epoxyenone system to the dienone system have long been known [82]. In addition, tylosin-related macrolides containing the epoxyenone system are being reported from fermentations, providing additional substrates for chemical modifications [150, 151]. 

To reduce or eliminate microbial attack, several methods can be used. They include freezing, drying, and the use of a preservative or a combination of any of the three. Commonly used preservatives include sorbic acid or sorbate, sodium benzoate, sodium salicylate, tylosin, formaldehyde, toluene, and thymol. The selection of a method or preservative will depend on the nature of the food, expected contamination, storage period and conditions, and the analyses that are to be performed. 

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