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HPLC-DAD and Fuorescence Determination of Veterinary Antibiotics from Wastewaters of Pharmaceutical Industry

Water is an essential and irreplaceable resource and should be carefully managed. In addition to its vital role in the biology of all living organisms, water is used for many other purposes in the modern world. For example, it plays a major role in industrial production, serves as a recipient for wastewater effluent and rainwater run-off, and is also used by man for shipping and leisure activities. Inevitably these anthropogenic activities can pollute water. The identification and monitoring of such pollutants requires powerful selective and sensitive analysis techniques. [1] Many veterinary pharmaceutical industries use the water as a recipient for wastewater effluent after treatment. Those wastewaters are usually loaded with very high concentration of pharmaceuticals, especially with veterinary antibiotics. The increasing use of antibiotics drugs in livestock, poultry production, and fish farming during the last five decades has caused a genetic selection of more harmful bacteria, which is a matter of great concern. Antibiotics are chemical substances produced by living organisms or derivates of these substances that suppress the growth of or kill other microorganisms in low concentrations. Nowadays the term antibiotics is extended to synthetic antibacterial agents as the sulphonamides or quinolones. [2] Antibiotics are some of the most produced and prescribed pharmaceuticals in last years. It is estimated that the 30% of total amount of antibiotics were used in the veterinary sector for therapeutic use or as growth promoters. Antibiotics are classified as emerging or new contaminants, or better as ignored environmental contaminants. This group of contaminants is manly composed of products used in large quantities in everyday life. Due to their physico-chemical properties, high water solubility and often poor degradability, they are able to penetrate through all natural filtration steps and man-made treatments. The characteristic of this group of contaminants is that they do not need to be persistent in the environment to cause negative effect since their high transformation/removal rates can be compensated by their continuous introduction into environment. Many classical wastewater treatment plants are not suitable for removing antibiotics from wastewaters of pharmaceutical industry, they passing sewage treatment plants, so, with the effluents can get into the receiving surface waters, from these reasons it is necessary monitoring the antibiotics in wastewaters before and after treatment. [3, 4] For this purpose it was necessary to develop a robust, simple, realistic and practical method capable of simultaneous determination of target compounds, hence HPLC diode array (DAD) and fluorescence (FLD) detector were the analytical techniques chosen. The pharmaceuticals investigated included three sulfonamides (sulfamethazine, sulfadiazine and sulfaguanidine), a sulfonamide synergist (trimethoprim), a tetracycline (oxytetracycline), a fluoroquinolone (enrofloxacine) and a beta-lactame (penicilline G / procaine). The selection of these compounds from the most widely used chemical classes of antibiotics with a range of physico-chemical properties ensured that studies were relevant. These groups of compounds have been identified as having high potential to reach the environment. The LC analyses were performed using a Varian ProStar 500 (Walnut Creek, California, USA) HPLC system consisting of a ProStar autosampler, ProStar 230 tertiary pump system, ProStar 330 diode array detector and ProStar 363 fluorescence detector, and thermostatted column compartment. A LiChrosphere 100 CN, 125 mm × 4.0 mm, particle size 5 μ m column (Merck, Darmstadt, Germany) was used to separate all compounds in the mixture. The mobile phase used in the chromatographic separation consisted of a binary mixture of solvents A (0.01 M oxalic acid) and B (acetonitrile). Simultaneous separations of studied pharmaceuticals were achieved at the following flow rate and mobile phase gradient program: the elution started with 100% of A which was maintained for 6 min, followed by a 19-min linear gradient to 50% of eluent B and 5-min linear gradient back to 100% of A. Flow rate started with 1.0 mL/min which was maintained for 6 min, followed by a 19-min linear gradient to 0.8 mL/min and 5-min linear gradient back to 1 mL/min. The investigated pharmaceuticals were eluted within 20 min. A 10-min post time allowed re-equilibration of the column. The separation was monitored at absorbance wavelength of 280 nm for DAD, and excitation 278 nm/emission 445 nm for all analytes by fluorescence detection. The column temperature was set to 30 °C. The injection volume was 20 μ L. The method involves pre-concentration and clean up by solid phase extraction (SPE) using Oasis HLB extraction cartridges. [5-9] The performance characteristics of the SPE-HPLC method were established by validation procedure. Selectivity, linearity, limits of detection (LOD) and quantification (LOQ), precision and recovery were studied. The described method was applied to the determination of the selected pharmaceuticals in wastewater from pharmaceutical industry.

high performance liquid chromatography; diode array detector; fluorescence detector; veterinary antibiotics; wastewater; pharmaceutical industry

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