Comparison of the Amicon Centrifree Micropartition System with the Sartorius SM 13249E Centrisart I Device to Determine Protein Free Phenytoin Concentrations

Abstract

The Amicon Centrifree micropartition system was compared with the Sartorius SM 13249E Centrisart I ultrafiltration device to determine non-protein-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 microM, 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 less than or equal to 5% of the free phenytoin concentration, whereas the Sartorius membranes absorbed 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.