Poster_metformin_knappe

antidiabetic Metformin University Medical Center Freiburg eMail: klaus.kuemmerer@uniklinik-freiburg.de Metformin is an oral anti-diabetic drug used for the treatment of diabetes mellitus type II. In Germany, 416 million defined daily doses (DDD) of Metformin were prescribed in 2007 (more than 830 tons), making it the most applied drug in terms of absolute dosage. With more than 40 years of usage, there has risen concern about its environmental fate. Metformin was not readily biodegraded in the Closed Bottle test (OECD 301D) within 30 days and was therefore tested in the Zahn-Wellenstest (OECD 302B) for its inherent biodegradability, where a partial elimination of the dissolved organic carbon (DOC) was found. To identify transformation products, chromatographic and mass spectrometric techniques (HPLC-MSn) were applied.
The Zahn-Wellens test (Fig. 1) assesses if an organic compound is aerobically biodegradable to anyextent. Test period was 30 days, start concentration 177.78 mg/L (corresponding 51.57 mg/L DOC). Samples of each day were measured upon their DOC and primary elimination by LC-MS/MS.
DOC analysis: Detaching particular carbon by centrifugating 30 min with 4000 rpm (Universal 32 R, Hettich Zentrifugen). Filtration with 25 mm PTFE syringe filter and cellulose nitrate membrane 45 µm (Rohr, Göttingen). TOC analyser (TOC 5000/ASI 5000 Version 3.10, Shimadzu).
Analysis of primary elimination: Chromatographic separation with a HPLC system (HPLC 1100 Agilent Technologies) on a modified reversed phase column. Mass spectrometric analysis with ESI-MSn (Esquire 3000 plus, Bruker Daltonics), positive ionization mode, scan range 20 m/z - 1000 m/z. DOC and LC-MS/MS (corresponding DOC) analysis of Metformin and Guanylurea
In both test vessels Metformin was not completely eliminated according to the DOC analysis (Fig. 2a). Results of the LC-MS analysis showed at test beginning only Metformin (Fig 3a & 3b) at tr,Met = 3.5 min, whereas from day 9 until test end Metformin almost completely disappeared and a new peak was found at tr,Gua = 3.0 min (Fig. 4a). This peak at m/z 103.4 was identified with MS/MS and MS3 (Fig. 4b) and by comparison with standards (same product ions and same retentiontime) as Guanylurea. Fig. 2b gives a proposal for the degradation path and fragmentation schemes. The test was valid sinceethylene glycol in the positive control vessel was eliminated to a degree of more than 70% within 14 days.
Metformin was completely eliminated in both test vessels of the Zahn-Wellens test, but not fully mineralised, leading to therecalcitrant substance Guanylurea. Due to the fact that Metformin isn‘t transformed by human metabolism, this microbial dead-end transformation product might have been present for decades in the aquatic environment without any knowledge of its existence nor properties. Analysis of environmental samples as well as toxicity assessment for Guanylurea are therefore under investigation.

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