The Amicon Centrifree micropartition system was compared with the Sartorius SM 13249E Centrisart I ultrafiltration device to determine nonprotein-bound phenytoin levels. Phenytoin was measured by an enzyme multiplied immunoassay technique (EMIT), adapted to a Cobas Bio centrifugal analyzer, and by a radiometric assay. From pooled human plasma with total phenytoin concentrations of 19.1, 44.0, and 95.1 μM, the following percentages of free phenytoin levels in the ultrafiltrate were obtained with Amicon: 10.6 ± 1.3, 10.5 ± 0.4, and 8.8 ± 0.2. The corresponding figures with Sartorius were 5.4 ± 0.8, 5.4 ± 0.5, and 4.9 ± 0.4, respectively (n = 5). Similar results were obtained with 3H-labeled phenytoin, ruling out the possibility that the discrepancies were due to interference with the EMIT assay. Phenytoin binding to the Amicon membrane was ≤5% of the free phenytoin concentration, whereas the Sartorius membranes adsorbed 80%. With the Amicon device, the free phenytoin concentration remained constant as serial ultrafiltrate volumes from the same sample were collected, whereas with Sartorius, free phenytoin increased from 2.5 ± 1.8% of the total concentration in the first fraction to 13.4 ± 1.6% in the last collection (n = 6). The results indicate that the Amicon Centrifree filter is an excellent tool to obtain protein-free phenytoin ultrafiltrates, whereas the Sartorius Centrisart I device is not suited for this purpose because of excessive adsorption of phenytoin.
Address correspondence and reprint requests to T. Zysset at Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, CH-3010 Berne, Switzerland.
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