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Determination Of Erythromycin And Its Degradation Products In Rivers Using Freeze Drying And Liquid Chromatography<dash>tandem Mass Spectrometry

Congress: 2015
Author(s): Kiana Kargar, John MacLachlan, Moyra McNaughtan, Joanne Roberts
Glasgow Caledonian University1

Keyword(s): Sub-theme 2: Surface water and groundwater,
AbstractIntroduction

Erythromycin A, a macrolide antibiotic, is produced by a strain of Saccharopolyspora erythraea (formerly Streptomyces erythreus)(Minas, 2005) and is widely used in both human and veterinary practice to treat bacterial infections against gram-positive bacteria (Kim et al., 2004). After consumption, erythromycin is concentrated in the liver and excreted in the bile. Less than 5% of the oral dose (12% to 15% IV) is excreted unchanged via urine into the sewage (Joint FAO/WHO Expert Committee on Food Additives et al., 2006). In wastewater treatment plants most macrolides (such as erythromycin) are only partially eliminated and residual amounts can reach surface waters or groundwater (McArdell et al., 2003). The accumulation and persistence of antibiotics in the environment can produce harmful effects such as an increase in antibiotic resistance in natural bacteria which could eventually lead to difficulties in treating infections in humans and animals (Hirsch et al., 1999). Erythromycin A is unstable in acidic and alkaline environments so it was also important to also identify its more stable degradation products that could be found in the environment. Knowing the concentration in ground and river water will help establish further studies on how to deal with the consequences of the presence of these antibiotics and their degradation products.

Methods

Erythromycin is most stable in deionised water. Deionised water was set to pH 3 using formic acid to prepare an acidic environment to degrade erythromycin and produce degradation products. These degradation products have characteristic LC-MS properties (retention time and mass ions) that are used to identify and quantify them alongside erythromycin.
Extraction methods for erythromycin (macrolide) and its degradation products from river water were developed, using Freeze Drying followed by High Performance Liquid Chromatography (HPLC) and Tandem Mass Spectrometry (MSn). It was important for the methods to be optimised to give the best recovery for erythromycin and its possible degradation products, taking into account that they may not be stable in real environmental conditions.
Freeze drying removes excess water and concentrates the final sample. It is already known that erythromycin is not volatile and recovery experiments are being carried out to determine this. HPLC analysis was performed on a Dionex Ultimate 3000 with Accucore C18 column (100x2.1mm) from Thermo Scientific and gradient elution. The composition of the mobile phase, gradient elution, flow rates and injection volume are listed in Table 1.

Table 1 HPLC conditions

Mass spectrometric detection was performed with Thermo Q Exactive operated in the positive ion mode and in high resolution.

Results

Degraded erythromycin produced more than 20 degradation products (peaks). Retentions times for erythromycin and its degradation products were achieved using the degraded acidic standards. Erythromycin A peak was detected at 6.3 minutes. Among the degradation products there are three that show to be more consistent and more stable than the others: erythromycin-C8H14O3 (dealkylation)-H2O (558 m/z) at 5.4 minutes, erythromycin-C8H14O3 (dealkylation) (576 m/z) at 4.8 minutes and erythromycin-H2O or anhydroerythromycin (716 m/z) at 8.8 minutes.
River samples were collected from upstream and downstream of a sewage treatment plant located along Breich Water, a substantial river of West Lothian. Samples were analysed using the developed method. LC-MS/MS was only able to detect small traces of erythromycin and anhydroerythromycin in downstream samples where none of the compounds listed in Table 2 were found in upstream samples. Erythromycin concentration was measured over a four day period from 30th June to 3rd July 2014.

Table 2 Measured erythromycin concentration in river water

Conclusion

An optimised method for analysis of erythromycin and its many degradation products was developed in the laboratory under acidic conditions. Three of the produced degradation products were shown to be more stable, and were identified and chosen to be monitored in river samples. River samples from upstream and downstream of a sewage treatment plant were monitored and traces of erythromycin and anhydroerythromycin were found in downstream samples and one upstream sample. Over a four day period erythromycin concentration was measured and first two days were double the following two. Monitoring the weather conditions (temperature and rainfall) on days that samples were taken showed a consistent dry period for all 4 days, suggesting that dilution due to rainfall is not a factor in different concentrations measured. This would suggest that the inconsistency is more related to human activity. This study also proved that erythromycin is not completely being eliminated in the sewage treatment plant. Further sampling will help to build erythromycin concentration profile over a 24 hour period in mentioned locations. 1. Hirsch, R., Ternes, T., Haberer, K., et al. (1999) Occurrence of antibiotics in the aquatic environment. The Science of the total environment [online], 225 (1-2): 109–18. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10028708

2. Joint FAO/WHO Expert Committee on Food Additives, Food and Agriculture Organization, World Health Organization, et al. (2006) Residue Evaluation of Certain Veterinary Drugs: Joint FAO/WHO Expert Committee on Food Additives, 66th Meeting 2006 [online]. Food & Agriculture Org. Available from: http://books.google.com/books?id=QNbUNjv70JMC&pgis=1 [Accessed 27 October 2014]

3. Kim, Y.-H., Heinze, T.M., Beger, R., et al. (2004) A kinetic study on the degradation of erythromycin A in aqueous solution. International Journal of Pharmaceutics [online], 271 (1-2): 63–76. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0378517303005969 [Accessed 11 November 2013]

4. McArdell, C.S., Molnar, E., Suter, M.J.F., et al. (2003) Occurrence and fate of macrolide antibiotics in wastewater treatment plants and in the Glatt Valley watershed, Switzerland. Environmental science & technology [online], 37 (24): 5479–86. Available from: http://www.ncbi.nlm.nih.gov/pubmed/14717154

5. Minas, W. (2005) Production of Erythromycin With Saccharopolyspora erythraea. Methods in Biotechnology [online], 18: 65–90. Available from: http://link.springer.com/protocol/10.1385%2F1-59259-847-1%3A065?LI=true

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