以HPLC/UV分析環境水體中Tetracycline及Quinolone類抗生素含量

Quantitative Analysis of Tetracycline and Quinolone Antibiotics in Aquatic Environment by Using HPLC/UV

莊舒雯(Shu-Wen Chuang)

指導教授 ： 洪慶宜

長榮大學/健康科學學院/職業安全與衛生研究所/碩士(2010年)

https://doi.org/10.6833/CJCU.2010.00006

摘要

近年來環境中抗生素抗藥性的議題相當受到重視，這可能與農畜業濫用抗生素有關。本研究結合固相萃取(SPE)與高效能液相層析儀紫外線偵測器(HPLC/UV)，建立一套適用檢測環境樣本中Nalidixic acid(NA)、Ciprofloxacin(CIP)及Tetracycline(TE)三種抗生素的分析方法。
水樣後經固相管柱萃取，通過逆相HPLC定量分析三種抗生素。採用Agilent HC-C18(2)管柱(150×4.6 mm, 5um)，移動相A為100% acetonitrile，移動相B為磷酸緩衝溶液。0-5分鐘移動A由30% acetonitrile改變為35%，5-10分鐘梯度維持35% acetonitrile。流速為1.0 mL/min，柱溫25oC，使用紫外光偵測器定量，抗生素Nalidixic acid、Ciprofloxacin及Tetracycline的最佳吸收波長分別為254nm、275nm及275nm，而時間則分別在8.3、1.9及2.3分鐘出現，檢量線之R2各為0.99996、0.99996及0.99973。以標準品推估，抗生素NA、CIP、TE的方法偵測極限分別為0.01、0.02、0.09 ppm。
本研究以純水中添加標準品測試C18、MCX及MAX等三種固相萃取匣的效能，其中NA以C18萃取匣有(100%)具最佳回收率； CIP及TE則以MCX具最佳萃取率(分別為105%及24%)。在畜牧廢水中添加NA、CIP及TE三種抗生素標準品至10ppm，平均回收率分別為47%、77%及14%；調整水樣的pH值為4，NA的回收率變高(100%)。
量測畜牧廢水未發現這三種抗生素，顯示廢水中抗生素的濃度低於分析方法的偵測極限。CIP使用MCX固相萃取匣回收率最佳；NA將水樣pH值調整為4，並使用C18固相萃取匣回收率最佳；而TE不適用本研究的方法。
因廢水中抗生素濃度低於大腸桿菌的最小抑制濃度，顯示畜牧廢水中微生物對人用抗生素的抗藥性可能係因畜牧預防性投藥，保留多重抗藥的基因所致。

關鍵字

抗生素 ； 畜牧廢水 ； 高效率液相層析儀

並列摘要

The issues of antimicrobial resistance spreading in environments have received much attention recently. It could be in connection with using too much antimicrobials agents in the animal husbandry. In the present study, a reliable method coupling solid phase extraction and HPLC/UV analysis was aimed to develop to simultaneously quantify Nalidixic acid(NA), Ciprofloxacin(CIP) and Tetracycline(TE) in the environmental samples.
After extraction and concentration by solid-phase extraction (SPE), the selected antibiotic compounds in samples were analyzed by a reverse-phase high performance liquid chromatography (HPLC). Antibiotics were separated by an Agilent HC-C18(2) column (150mm×4.6mm, 5µm) with a gradient elution at a flow rate of 1.0 mL/min. Eluent A was acetonitrile (ACN) and eluent B was 0.2% phosphoric buffer. The concentration of the mobile phase A was changed from 30% to 35% in the first 5 minute, then maintained 35% ACN for another 5 minute. Column temperature was set at 25 oC. NA, CIP and TE were quantified by a Diode Array Detector at UV absorbance of 254, 275 and 275 nm, at retention time of 8.3, 1.9, and 2.3 min with a correlation coefficient (R2) of 0.99996, 0.99996, and 0.99973, respectively. The method detection limits, estimated by standard solutions, of NA, CIP and TE were 0.01, 0.02 and 0.09 ppm, respectively.
The effectiveness of the three SPE cartridges was tested by spiking antibiotic standards in ultra-pure water and a typical swine wastewater. The results of antibiotic-spiked water experiment indicated the most suitable SPE cartridge for NA was C18 (100% recovery), while MCX was the best SPE cartridge for both CIP and TE with recovery of 105% and 24%, respectively. Using these antibiotic SPE combinations, the recoveries of spiked standards in the wastewater were 47%, 77%, and 14% for NA, CIP and TE, respectively. Adjusting pH to 4, the recoveries of the NA from wastewater enhanced to 100%.
The concentrations of NA, CIP and TE in the swine wastewater, however, were non-detectable. The study concluded that the most suitable SPE for NA is C18 after acidification to pH=4. The most suitable SPE for CIP is MCX, while none of the three cartridges can be used to concentrate TE.
Because the concentration of the three antibiotics was lower than the maximum inhibitory concentration, the present of bacteria resistant to the human-used antimicrobial agents might be resulted from the preservation of multiple resistant gene by the use of prophylactic drags in the animal husbandry.

並列關鍵字

antibiotics ； swine wastewater ； high performance liquid chromatography

參考文獻

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