Isolation and structure characterization of related impurities in etimicin intermediate P1 by LC/ESI–MSn and NMR

• Related impurities of etimicin key intermediate P1 were detected by HPLC methods.
• Four major related impurities were isolated from P1 by column chromatograpy.
• Their structures were established by means of LC–MSn and NMR techniques.
• Three impurities are new compounds and all NMR data were first reported.
• The possible mechanism for the formation of these impurities is discussed.

Etimicin intermediate 3,2″,6″-N,N,N-triacetyl gentamicin C1a (P1), is a key intermediate of etimicin, which is a semi-synthetic aminoglycoside antibiotic effective to both gram-positive and gram-negative bacteria infections. Four major related impurities of P1 were detected by HPLC–ELSD and ESI–MSn methods. Weakly acidic cation exchange resin, CM-sephadex and silica gel column chromatography were used for the isolation and purification of four major impurities. By means of ESI–MSn and NMR analysis, related impurities were characterized as 3,2″-N,N-diacetyl gentamicin C1a (1), 3,2″,6″-N,N,N-triacetyl gentamicin C2b (2), 2″-N-acetyl gentamicin C1a (3), and 2″,6″-N,N-diacetyl gentamicin C1a (4). Impurities 1, 2, 4 are novel compounds and the NMR data of these isolates were first reported in this paper. The possible mechanism for the formation of these impurities is also discussed.

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