Comparison of the Beckman TL-100 Ultracentrifuge with the Amicon Centrifree Micropartition Filters to Determine Protein-Free Phenytoin Concentrations

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Abstract
Non-protein-bound phenytoin levels obtained by ultracentrifugation (UC) with the Beckman TL-100 desk-top ultracentrifuge were compared with those obtained by ultrafiltration (UF) using the Amicon Centrifree Micropartition device. Total phenytoin concentrations were measured by an enzyme multiplied immunoassay technique (EMIT) adapted to a Cobas Bio centrifugal analyzer, whereas free levels were estimated by a radiometric assay. The following conditions for UC were established: 350,000 g for 1 h, sample volume 250 .mu.l. From three pooled human plasmas containing 18.8, 44.4, and 73.9 .mu.M total phenytoin, the following percentages of free phenytoin levels were obtained with UF: 10.4 .+-. 0.1 (SD), 10.7 .+-. 0.6, and 11.1 .+-. 0.1%. The corresponding figures with UC were 10.8 .+-. 0.9, 11.4 .+-. 0.4, and 12.0 .+-. 0.6%, respectively. Within-sample precision showed a coefficient of variation between 3.5 and 8.0% for UC, with UF corresponding values between 1.0 and 5.6%. Free phenytoin levels in 19 patients undergoing phenytoin therapy were between 6.8 and 19.3% with UF (mean 10.1 .+-. 3.0%) and 8.7 and 23.9% (mean 11.9 .+-. 3.5%; p < 0.001 by paired t test) with UC. Least-squares linear regression analysis resulted in the following equation: percentage free phenytoin by UC = 1.11 .times. % free phenytoin by UF.sbd.0.70 (r = 0.935, p < 0.001). The results indicate that within certain reservations UC under the conditions tested may be an alternative to UF or equilibrium dialysis.