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Abstract

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.

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