Tylosin analysis

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). 

Figure 9 HPLC chromatograms of culture broth of S. fradiae MBBF-c (A) and S. fradiae MBBF-c(pABllAEH) (B, C). In the fermentation at (C), L-leucine was fed into the culture, and the supernatant was diluted by 20-fold with acetonitrile for analysis. Abbreviations I, tylosin VI, 3-O-acetyltylosin VII, 3,4"-di-0-acetyltylosin VIII, 3-(9-acetyl-4"-O-propionyl tylosin IX, 3-0-accty 1 -4 O- b ut y ry I ty I o si n X, 3-0-acetyl-4"-0-isovalerylty-losin (AIV). (From Ref. 22.)...

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. 

According to this modular analysis, each protein catalyses two cycles of chain extension. The term cassette has been proposed for the giant proteins [34]. All three cassettes in the erythromycin cluster are bimodular, but in other systems, such as the rapamycin [35] and tylosin [36] PKSs, the size of a cassette can vary from one to six chain extension modules. The three cassettes co-operate in some way to form an extraordinarily complex molecular assembly hne. The biosynthetic intermediates remain PKS-bound throughout the whole synthetic sequence via thioester links. A challenging feature of this organisation is the mechanism which controls the ordering of the cassettes in the assembly Hne so that the transfer of the growing chain from one cassette to the next is correctly controlled. 

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.

Merson-Davies, L. A., and Cundliffe, E. (1994). Analysis of five tylosin biosynthetic genes from the tyllBA region of the Streptomyces fradiae genome. Mol. Microbiol. 13, 349-355. 

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%. 

Thompson TS, Pernal SF, Noot DK, Melathopoulos AP, van den Heever JP, Degradation of incurred tylosin to desmycosin-rmplications for residue analysis of honey. Anal. Chim. Acta 2007 586(l-2) 304-311. 

---