Journal of Pharmaceutical and Biomedical Analysis 34 (2004) 559–567 Isolation, structural elucidation and in vitro activity of 2-acetyl-2-decarboxamido-oxytetracycline against environmental relevant bacteria, including tetracycline-resistant bacteria Anne Kruse Lykkeberg a,∗ , Gitte Sengeløv b , Claus Cornett a , Jette Tjørnelund a , Steen Honoré Hansen a , Bent Halling-Sørensen a a Department of Analytical Chemistry, The Danish University of Pharmaceutical Science, Universitetsparken 2, DK-2100 Copenhagen, Denmark b Department of Microbiology, Danish Veterinary Laboratory, Bülowsvej 27, DK-1790 Copenhagen, Denmark Received 25 June 2003; received in revised form 20 October 2003; accepted 23 October 2003 Abstract 2-Acetyl-2-decarboxamido-oxytetracycline (ADOTC) is a major impurity of oxytetracycline (OTC) produced as a side product during fermentation. ADOTC was isolated from OTC and other impurities using preparative HPLC. The preparative column was an Xterra MS, C 18 chromatographic column (100 mm × 19 mm i.d., 5 m), and the mobile phase contained methanol–water (27:73 (v/v)) with 0.08 M formic acid added. The flow rate was 9.0 ml/min. It was possible to isolate few milligram ADOTC in a day. The compound was unambiguously identified using NMR and MS–MS. The anti-microbial activity against activated sludge bacteria was determined giving a potency of only 3% of that of OTC. With tetracycline-resistant bacteria, no anti-microbial activity was observed, indicating a mode of action similar to that of OTC. © 2003 Elsevier B.V. All rights reserved. Keywords: 2-Acetyl-2-decarboxamido-oxytetracycline; Preparative HPLC; NMR; Tandem mass spectrometry; Microbial testing; Resistance 1. Introduction 2-Acetyl-2-decarboxamido-oxytetracycline (ADO- TC) is a side product of oxytetracycline (OTC) pro- duced by Streptomyces rimosus. It is, therefore, found as an impurity in the raw material of OTC [1] and will ∗ Corresponding author. Tel.: +45-35-30-60-00/64-63; fax: +45-35-30-60-10. E-mail address: ak@dfh.dk (A.K. Lykkeberg). be present in formulations of OTC including veteri- nary medicine. ADOTC is also expected to be present along with OTC in urine and faeces from treated patients and animals. ADOTC is mentioned as an im- purity of OTC in the European Pharmacopoeia (Ph. Eur.) and the content of ADOTC has been limited to 2% of OTC. Other tetracyclines contain correspond- ingly 2-acetyl-2-decarboxamido-impurities as well [2]. The structures of OTC and ADOTC are shown in Fig. 1. 0731-7085/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0731-7085(03)00629-0