Determination of free levels of phenytoin in human plasma by liquid chromatography/tandem mass spectrometry

A liquid chromatography combined with tandem mass spectrometry assay for th e determination of free levels of the highly protein bound drug phenytoin ( 5,5-diphenylhydantoin) in human plasma is described. The assay was demonstr ated to be reliable, accurate and precise, and specific for phenytoin. The procedure involves isolation of the unbound drug from the drug/protein comp lex by ultrafiltration. Liquid-liquid extraction was employed to extract th e resultant ultrafiltrate. PHT was separated on a 50 x 3 mm reversed-phase column using isocratic mobile phase conditions that yielded a run time of 1 .5 min, enabling high throughput sample analysis. Linearity was obtained ov er the range 5.00 to 500 ng/ml, Both between-run and within-run coefficient s of variation were less than 15% and accuracy's across the assay range wer e all within 100+/-10%. The assay was successfully implemented to support a clinical interaction study with phenytoin. (C) 2000 Elsevier Science B.V. All rights reserved.