Journal of Chromatography A, 1509 (2017) 60–68 Contents lists available at ScienceDirect Journal of Chromatography A jo ur nal ho me pag e: www.elsevier.com/locate/chroma Full length article Comprehensive determination of macrolide antibiotics, their synthesis intermediates and transformation products in wastewater efﬂuents and ambient waters by liquid chromatography–tandem mass spectrometry Ivan Senta, Ivona Krizman-Matasic, Senka Terzic ∗ , Marijan Ahel Division for Marine and Environmental Research, Rudjer Boskovic Institute, Bijenicka c. 54, 10000 Zagreb, Croatia a r t i c l e i n f o Article history: Received 15 February 2017 Received in revised form 31 May 2017 Accepted 1 June 2017 Available online 3 June 2017 Keywords: Antibiotics Azithromycin Erythromycin Clarithromycin Transformation products Liquid chromatography–tandem mass spectrometry Wastewater Surface water a b s t r a c t Macrolide antibiotics are a prominent group of emerging contaminants frequently found in wastewater efﬂuents and wastewater-impacted aquatic environments. In this work, a novel analytical method for simultaneous determination of parent macrolide antibiotics (azithromycin, erythromycin, clarithromycin and roxithromycin), along with their synthesis intermediates, byproducts, metabolites and transforma- tion products in wastewater and surface water was developed and validated. Samples were enriched using solid-phase extraction on Oasis HLB cartridges and analyzed by reversed-phase liquid chromatog- raphy coupled to electrospray ionization tandem mass spectrometry. The target macrolide compounds were separated on an ACE C18 PFP column and detected using multiple reaction monitoring in positive ionization polarity. The optimized method, which included an additional extract clean-up on strong anion-exchange cartridges (SAX), resulted in high recoveries and accuracies, low matrix effects and improved chromatographic separation of the target compounds, even in highly complex matrices, such as raw wastewater. The developed method was applied to the analysis of macrolide compounds in wastew- ater and river water samples from Croatia. In addition to parent antibiotics, several previously unreported macrolide transformation products and/or synthesis intermediates were detected in municipal wastewa- ter, some of them reaching g/L levels. Moreover, extremely high concentrations of macrolides up to mg/L level were found in pharmaceutical industry efﬂuents, indicating possible importance of this source to the total loads into ambient waters. The results revealed a signiﬁcant contribution of synthesis intermediates and transformation products to the overall mass balance of macrolides in the aquatic environment. © 2017 Published by Elsevier B.V. 1. Introduction In the last 20 years, a large number of studies [1–5] reported on widespread occurrence of antimicrobial compounds in wastewater and in the aquatic environment. Since the spread of antibiotic resis- tance is considered to be one of the most serious global threats to human health in the upcoming decades [6], the ubiquitous presence of antibiotic residues in the environment raised a great concern about possible contribution of the aquatic route to proliferation of the resistant bacterial strains [7]. In order to meet the needs of reliable and sensitive assessment of antibiotic exposure concen- trations in different matrices, a number of analytical methods for the determination of antimicrobials in environmental samples have ∗ Corresponding author. E-mail address: terzic@irb.hr (S. Terzic). been described in the literature over the past decade. Many of the published methods are multiresidual and multiple-class methods, which typically include a smaller number of selected representa- tives from each antimicrobial class [8,9]. Moreover, the majority of the published methods have focused exclusively on the parent com- pounds [5], or, at best, include some major metabolites [10,11]. As a consequence, the contribution of transformation products (TPs), as well as synthesis intermediates and byproducts, to the total mass loads of antimicrobial-related compounds in the environment is much less understood. Macrolide antibiotics represent one of the most prominent classes of antimicrobial agents, with widespread usage in both human and veterinary medicine. In the global antibiotic consump- tion in 2010, macrolides were ranked third [12], and their usage in human medicine in Croatia and other European countries [13] is in accordance with the global ﬁgures. Moreover, together with http://dx.doi.org/10.1016/j.chroma.2017.06.005 0021-9673/© 2017 Published by Elsevier B.V.