Semduramicin analysis

Instruments. The HPLC system for analysis of unchanged semduramicin was modeled after an LC-post column reaction system described earlier for salinomycin sodium in feeds (15-16). The mobile phase consisting of ethyl acetate, iso-octane, glacial acetic acid, triethylamine, methanol (650 + 350 + 44-2 + 1) was used in conjunction with a Dupont Zorbax silica 4.6 mm X 25 cm analytical column. NMR spectra were recorded on a Bruker WM-250 spectrometer (modified to incorporate a pulse programmer and Aspect-3000 data system) and a Bruker AM-500 spectrometer, using 50 mg samples dissolved in CDCI3. The 13C and IH chemical shifts for semduramicin were assigned as reported elsewhere (17). FAB (fast atom bombardment) mass spectra were obtained on a VG 70/250 S spectrometer at 1000 resolution. 

Spectral Identiflcation of Metabolites and F . Interpretation of the results of mass spectrometry of semduramicin sodium and its metabolites was facilitated by comparison with the fast atom bombardment mass spectral (FAB-MS) analysis of maduramicin sodium and other iono-phores (23), the thermospray liquid chromatography/tandem mass spectrometry of maduramicin and its metabolites (14, 24), and the extensive review of the mass spectrometry of polyether antibiotics (25). In this way, mass spectral interpretations were formulated for semduramicin and... 

Analysis of the IH-NMR spectrum of metabolite E showed only a singlet at 3.52 ppm suggesting loss of the ether linked methyl at position 44. This was corroborated by observed changes in the chemical shift for protons in positions 41 and 42, but none for the proton in position 6 it remained unchanged relative to semduramicin (apparent triplet at 3.11 ppm). 

Likewise, analysis of the spectrum of metabolite "F showed the methyl singlet at 3.36 ppm intact suggesting loss of the ether linked methyl at position 45. Loss of the apparent triplet at 3.11 ppm (proton in position 6 of semduramicin) and the presence of a seemingly unchanged multiplet at 2.81 ppm (similar to proton in position 41 of semduramicin) supported this interpretation. Thus, the structures postulated for metabolites E and F by mass spectrometric analysis were confirmed by IH NMR. 

As defined by reverse isotope dilution analysis and metabolic profiling experiments, unchanged semduramicin sodium was the major component of total residues in liver at the 6 hour withdrawal period. At later withdrawal times, unchanged semduramicin sodium levels declined by a biphasic pattern in liver similar to that of total residues. 

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