Quantification of Free Phenytoin by Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS)

  • Judy Peat
  • Clint Frazee
  • Uttam GargEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1383)


Phenytoin (diphenylhydantoin) is an anticonvulsant drug that has been used for decades for the treatment of many types of seizures. The drug is highly protein bound and measurement of free-active form of the drug is warranted particularly in patients with conditions that can affect drug protein binding. Here, we describe a LC/MS/MS method for the measurement of free phenytoin. Free drug is separated by ultrafiltration of serum or plasma. Ultrafiltrate is treated with acetonitrile containing internal standard phenytoin d-10 to precipitate proteins. The mixture is centrifuged and supernatant is injected onto LC-MS-MS, and analyzed using multiple reaction monitoring. This method is linear from 0.1 to 4.0 μg/mL and does not demonstrate any significant ion suppression or enhancement.

Key words

Phenytoin Free phenytoin Mass spectrometry Seizures Convulsions Epilepsy 


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Pathology and Laboratory MedicineChildren’s Mercy Hospitals and ClinicsKansas CityUSA

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