中華絨螯蟹血清與肌肉中Oxolinic acid殘留檢測   全文下載

Development of a Technique to Detect the Residue of Oxolinic Acid in the Serum and Muscle of Chinese Mitten Crab, Eriocheir Sinensis

本研究擬開發以高效液相層析法搭配螢光檢測器，用來檢測中華絨螯蟹血清與肌肉中之oxolinic acid殘留量。先將oxolinic acid溶於methanol/NaOH製成標準品，以0.05 M PBS（pH 7.4）定量稀釋後，分別加入蟹體0.5 mL之血清和0.5 g已均質化之肌肉，再分別經超音波水浴震盪後離心。將血清上清液及經hexane去脂後之肌肉水層液分別通過C-18固相萃取（SPE）管柱，最後將管柱中結合藥物以methanol/NH4OH（75: 25 v/v）沖提出並以氮氣吹乾後進行高效液相層析。本研究所開發出之檢測技術條件為：Cosmosil 5C18-MS（5 μm, 4.6 × 150 mm i.d.）管柱，以25 : 75之acetonitrile/0.02 M PBS（pH 3）為移動相，流速為1 mL/min，螢光檢測器之激發波長為335 nm，散發波長為378 nm。檢測結果顯示約在8分鐘可出現一明顯之oxolinic acid吸收波峰，其波峰面積回歸曲線係數高於0.999，檢測極限可達到1 μg L-1，以100 ppb oxolinic acid添加於血清與肌肉之回收率分別為92%與82%。因為無法於市售中華絨螯蟹體內偵測到殘留，所以分析經餵食oxolinic acid 7天後之螃蟹樣本，結果顯示本研究所用之方法可以自該蟹體血清與肌肉中檢測出oxolinic acid殘留。由此可知我們所開發之技術能應用於中華絨螯蟹組織中oxolinic acid之殘留檢測。"

We analyzed the residual of oxolinic acid in the serum and muscle of the Chinese mitten crab by high-performance liquid chromatography (HPLC) coupled with a fluorescence detector. A standard solution of oxolinic acid was prepared by dilution with 0.05 M phosphate buffered saline (PBS) (pH 7.4). Serum and muscle samples were completely homogenized prior to addition of oxolinic acid. Samples were then transferred into an ultrasonic water bath and centrifuged. The supernatants of serum and water layer of hexane defatted muscle were passed through C-18 solid phase extraction columns. Finally, oxolinic acid in the column was eluted with methanol/NH4OH (75:25 v/v), and then was evaporated to dry by nitrogen gas. Extracted oxolinic acid was determined by HPLC with a fluorescence detector (excitation/emission = 335 nm/378 nm) and a Cosmosil 5C18-MS reverse phase column (5 μm, 4.6 × 150 mm i.d.). A mixture of acetonitrile and 0.02 M PBS pH 3.0 (25:75) was used as the mobile phase at a flow rate of 1.0 mL/min. A peak with retention time around 8 min was observed. With the addition of 100 μg L-1 oxolinic acid treatments, the mean recoveries from serum and muscle were 92% and 82%, respectively. The detection limit was 1 ppb and standard curves spiked at different levels showed a good linear correlation coefficient (R2 > 0.999 for oxolinic acid). Because residual oxolinic acid could not be detected in all tested crabs initially, five crabs were fed with oxolinic acid for 7 days by oral administration on purpose and the residual oxolinic acid in both sera and muscle were measured, respectively. The results showed that the residual oxoilinic acid in both sera and muscle could be quantified by this method. Therefore, our developed technique may be employed as a practical method for detection of residual oxolinic acid residue in Chinese mitten crabs or other aquaculture products.