Figure 6-5. HPLC chromatogram for catfish AT18 intestine. Rep 2 refers to second
 sample taken from AT18 intestine.


Final Method

 Liver tissue, 0.1 g, or 0.2 g intestinal mucosa were placed at the bottom of a small

thick glass homogenizing tube (Thomas AA717). Using a rough tipped pestle attached to

an electric drill, the tissue was homogenized for 10 seconds with 0.4 or 0.3 mL (liver or

intestine respectively) of 0.3 M NH4H2PO4. The tube in the homogenate was then placed

in a briskly boiling water bath for 1 minute after which it was removed and placed on ice.

The boiled mixture was briefly rehomogenized. The tubes were centrifuged, and the

supernatant (still containing suspended matter) was transferred into a 1.5 mL microfuge

tube and recentrifuged for 15 minutes at 16,000g. The supernatant was then filtered by

spin-centrifugation (using 0.45 CIM Spin-filters (Costar, Corning Inc., NY)) at 16,000g

for 5 minutes. A sample, 50C1L, was analyzed by HPLC (Model 2300 pump (ISCO,

Lincoln, NE) with Dynamax UV absorbance detector (Rainin, Woburn, MA)).