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

J Pharm Biomed Anal. 2000 Aug 15;23(2-3):573-9. doi: 10.1016/s0731-7085(00)00338-1.


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.

MeSH terms

  • Anticonvulsants / blood*
  • Chromatography, Liquid / methods*
  • Hemolysis
  • Humans
  • Mass Spectrometry / methods*
  • Phenytoin / blood*
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Anticonvulsants
  • Phenytoin