Due to a global problem of growing bacterial resistance, discovery of new antibiotics is of an outmost importance and various studies are in progress to overcome the resistance mechanisms.[1- 3] Macrozones are novel bioactive conjugates of azithromycin and thiosemicarbazides that posses very good in vitro antibacterial activity against selected Gram-positive and Gram-negative bacteria. [4] Efficient and rapid purification and isolation of newly synthesized bioactive compounds is a crucial step prior to biological evaluation. Furthermore, isolation and identification of impurities is very important procedure during the drug development in pharmaceutical industry. Preparative and semipreparative HPLC techniques are classical purification methods, but they can be time and solvent consuming. In this study we have successfully used hyphenated LC-SPE/NMR approach for the isolation and structural characterization of main components and some impurities in 9a- and 4''- tetrahydrofurfuryl macrozone reaction mixtures. Structures of the isolated compounds were proposed on the basis of the analysed one- and two-dimensional NMR spectra and MS spectra, respectively.