Determination of antibiotics in different water compartments via liquid chromatography–electrospray tandem mass spectrometry

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Abstract

For the determination of 18 antibiotics in water samples down to the lower ng/l range, an analytical multi method is presented. The analytes belong to different groups of antibiotics such as penicillins, tetracyclines, sulfonamides and macrolid antibiotics. Samples were enriched using a universal freeze–drying procedure or a solid-phase extraction facultatively. Analysis was performed by liquid chromatography with electrospray–tandem MS detection. Chromatography required different columns and eluents. Mean recovery rates were in excess of 70%, however, with one exception and a quantitation limit of 50 ng/l for the tetracyclines and 20 ng/l for all other antibiotics were set.

Introduction

One of the most relevant topics in today's environmental analytical chemistry is water quality. In order to characterize water quality, concentrations of polar organic pollutants have to be determined. In addition to pesticides, industrial chemicals and their metabolites, pharmaceutical substances have experienced a fast growing interest. Recent studies have shown that a multitude of drugs are present in aquatic systems 1, 2, 3. The interest in the analysis of antibiotic residues in the environment arises from the fact that they are suspected of being responsible for the appearance of bacterial strains that are resistant to antibiotics which are important drugs for the treatment of many serious infections.

In Germany the annual production of antibiotics is in the range of 2000 tons per year. One such main group are the penicillins, with production rates in Germany of approximately 900 tons/year [4]. Tetracyclines, sulfonamides and macrolid antibiotics are some additional important groups of antibiotics for which a proper analytical method in water down to the ng/l range has to be designed (see Fig. 1). Intake pathways into the aquatic environment result from their applications in both human and veterinary medicine. After consumption the active compounds are often metabolized only partially and are then excreted via urine or feces [5]. Thus, they are presumably present in raw sewage and may even leave the sewage treatment plants (STPs) as they have probably not been fully eliminated 2, 3, 6.

The use of those substances as feed additives in veterinary medicine underlies only minor legal regimentation. Application amounts are not available. Therefore, no estimation for the environmental intake of these substances can be made. As these compounds are most likely to be found at very low concentrations in the different compartments of the aquatic environment, like STP influents and effluents, surface, ground and drinking water 7, 8, it is necessary to develop a sensitive procedure which has the capability to confirm the presence of antibiotics down to the lower ng/l range.

Most of the continuously monitored water contaminants are determined via gas chromatography–mass spectrometry (GC–MS). A separation of the antibiotics mentioned herein via GC requires derivatization of the polar moieties. In literature some methods for the determination of e.g., chloramphenicol and sulfamethazine via GC–MS have been described 9, 10. However, as the analyte groups show different properties concerning number and kind of functional groups (see Fig. 1), it seems quite difficult to develop a universal derivatization procedure suitable for all the substances. High-performance liquid chromatography (HPLC) coupled to a tandem MS detector is a powerful technique for separation, identification and quantitation of polar compounds and was therefore chosen as a preferable method. As, macrolid antibiotics, which are made up of three structure elements, the erythronolide aglycone and two sugar moieties (desosamine and cladinose as described in Fig. 1), show no measurable UV absorption in the spectrum of conventional deuterium lamps, most publications concerning the determination of these substances describe electrochemical detection [11]. Generally, most of the published methods concerning the determination of antibiotic drugs are designed for complex matrices like meat, milk, blood etc., and achieve only relatively high detection limits in the range of several hundred ng/kg or ng/l, respectively 12, 13, 14, 15, 16. In addition most of the applied extraction procedures are especially designed for only one special class of antibiotics e.g., tetracyclines.

The aim of this work was to establish an effective and simple multi-method in order to determine the amounts of several antibiotic substances via LC–MS–MS down to the lower ng/l range.

Section snippets

Experimental

Reference compounds were purchased from Sigma except for roxithromycin and clarithromycin which were supplied courtesy of the manufacturers (Roussel Uclaf, France and Abbott, Germany). Standard solutions were stored in phosphate buffer (see below) at −20°C. They were always renewed after two months. All solvents were utilized in gradient grade or higher quality.

Results and discussion

Due to the predicted and previously detected low concentrations of antibiotics in the aquatic environment, a preconcentration step was necessary prior to measurement. Therefore, two alternative extraction methods, a freeze-drying and a SPE method, have been developed and tested with regard to their suitability for the enrichment of antibiotics (see below). The obtained extracts were then separated via HPLC using three different gradients and columns according to the three analyte groups as

Conclusions

LC–electrospray tandem MS is a powerful analytical technique for the sensitive determination of polar micro contaminants in water like antibiotics. The method presented herein offers a simple, effective and sensitive possibility to quantify 18 antibiotics down to the lower ng/l range in different water matrices. Lyophilization as a universal preconcentration step allows an easy extension of the method to the analysis of various other relevant compounds.

Five of the examined drugs present in a

Acknowledgements

This work was supported by grants of the German Federal Ministry for Education and Research (BMBF) and the Hessian Ministry for Environment, Energy, Youth, Family and Health.

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