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 the determination of free levels of the highly protein bound drug phenytoin (5,5-diphenylhydantoin) in human plasma is described. The assay was demonstrated to be reliable, accurate and precise, and specific for phenytoin. The procedure involves isolation of the unbound drug from the drug/protein complex by ultrafiltration. Liquid-liquid extraction was employed to extract the 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 over the range 5.00 to 500 ng/ml. Both between-run and within-run coefficients of variation were less than 15% and accuracy's across the assay range were all within 100 +/- 10%. The assay was successfully implemented to support a clinical interaction study with phenytoin.
Bardin
S
S
Searle, Skokie, IL 60077, USA.
Ottinger
J C
JC
Breau
A P
AP
O'Shea
T J
TJ
D000927:Anticonvulsants / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006461:Hemolysis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010672:Phenytoin / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000927:Anticonvulsants; D010672:Phenytoin