Improved thin-layer chromatographic detection of diethylstilbestrol and zeranol in plasma and tissues isolated with alumina and ion-exchange membrane columns in tandem.

Clean-up procedures for the isolation of zeranol and diethylstilbestrol (DES) were modified to reduce the analysis time and to increase the efficiency of purification. Several dyes (Fast Blue BB, Fast Corinth V, Fast Blue RR, Fast Blue B, Fast Red Violet B and Fast Violet B) were evaluated, and their minimum detectabilities were determined. Conditions for non-instrumental, semi-quantitative thin-layer chromatography were optimized. Zeranol and DES in plasma and tissues were determined using modified procedures. Enzyme digestion brought about significant improvement in detectabilities of zeranol and DES in both fortified and incurred plasma, serum and tissues. Minimum detectabilities for zeranol and DES were 25 ppb in fortified plasma and tissues. The amount of incurred zeranol measured in the serum of an experimental cow was increased four times, i.e. from 50 to 200 ppb, after protease digestion. Glucuronidase digestion showed an eight-fold increase in detection of incurred zeranol levels in bovine liver eight times. These results suggest that digestion releases zeranol and DES from protein and glucuronide complexes, thereby allowing detection of low levels of zeranol and DES which may not be detectable without digestion. Further modification of the purification with an ion-exchange membrane reduced the analysis time by 25%, and the membranes were regenerated up to ten times without loss of activity, allowing an automated process. This method utilizes inexpensive equipment and avoids use of organic solvent, in this case diethyl ether.