Journal of Pharmaceutical and Biomedical Analysis 158 (2018) 47–53 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis j o ur na l ho mepage: www.elsevier.com/locate/jpba pH-Dependent stability of azithromycin in aqueous solution and structure identification of two new degradation products Maria Grazia Saita a , Danilo Aleo a , Barbara Melilli a , Sergio Mangiafico a , Melina Cro a , Claudia Sanfilippo b , Angela Patti b,∗ a MEDIVIS – Corso Italia, 171-95127, Catania, Italy b CNR – Istituto di Chimica Biomolecolare, Via Paolo Gaifami 18, I-95126, Catania, Italy a r t i c l e i n f o Article history: Received 29 March 2018 Received in revised form 25 May 2018 Accepted 26 May 2018 Available online 28 May 2018 Keywords: Azithromycin Aqueous solution Lactone opening pH-Dependent degradation HPLC analysis a b s t r a c t The degradation profile of azithromycin in buffered solutions was investigated using HPLC and found to be pH dependent in the range of 6.0–7.2. Desosaminylazitromycin, derived from hydrolytic loss of cladi- nose of the parent molecule, was the major degradation product at pH 6.0 but its amount progressively decreased moving toward pH 7.2. Two additional unreported degradation products were also observed and their structures were fully elucidated by MS- and NMR-spectroscopy to be associated with opening of the macrocyclic lactone ring. © 2018 Published by Elsevier B.V. 1. Introduction Azithromycin (1) is a semi-synthetic macrolide antibiotic with a broad spectrum of activity against a variety of Gram-positive and Gram-negative bacteria. Azithromycin has been widely used since mid 80 ′ s in clinical treatment of respiratory infections and sexually transmitted diseases [1]. More recently, nonantimicrobial effects and immunomodulatory activity have also been evidenced for this drug expanding its use in the long-term therapy of chronic lung pathologies [2]. Azithromycin is classified as an azalide, a subclass of macrolides, and it is structurally characterized by a 15- membered macrocyclic lactone ring bearing two sugar residues as substituents in the 3- and 5-positions. Although its total synthesis has been recently reported [3], compound 1 is mainly produced from chemical modification of erythromycin [4–6], through the insertion of a methyl-substituted nitrogen in place of a carbonyl group in the parent molecule with simultaneous expansion of its 14-membered lactone ring, ulti- mately resulting in higher acid stability [7,8], enhanced biological activity and better bioavailability than erythromycin [9]. Impuri- ties deriving from the erythromycin fermentation process and/or subsequent chemical reactions can be found in bulk samples of ∗ Corresponding author. E-mail address: angela.patti@cnr.it (A. Patti). azithromycin along with degradation products [10]. Due to the importance of understanding the impurity profiles of pharmaceu- ticals [11], different HPLC methods with various detectors [12–15] were developed to analyze 1 for manufacturing quality monitor- ing, formulation stability evaluation, pharmacokinetic or metabolic profiling. HPLC–MS techniques proved to be effective in simultane- ous detection and identification of azithromycin metabolites and degradation products [16]. The stability of 1 has been investigated in different condi- tions and sensitivity to pH-variations and light exposure has been reported [17–19]. In a study on the chemical stability of 1 in buffered aqueous solution [20], pH 6.3 was shown optimal and desosaminylazitromycin 2, also known as impurity J [21], deriv- ing from the hydrolytic loss of cladinose sugar, was reported as the major degradation product in the 4.0–7.2 pH range. During the process of developing a quasi neutral aqueous oph- thalmic formulation containing 1 the degradation profiles of 1 in aqueous solutions with pH ranged from 6.0 to 7.2 were further investigated and the formation of additional two new degradation products, 3 (named impurity X) and 4 (named impurity Y), besides 2 was observed. The structures of 3 and 4 were fully elucidated using MS and NMR analyses. https://doi.org/10.1016/j.jpba.2018.05.042 0731-7085/© 2018 Published by Elsevier B.V.