A Simple Isotope Dilution Electrospray Ionization Tandem Mass Spectrometry Method for the Determination of Free Phenytoin

Abstract

Phenytoin (diphenylhydantoin) is an anticonvulsant drug frequently prescribed for the treatment of many types of seizures. Because the drug is highly protein bound (90%-95%) and many conditions can displace the drug from proteins, the measurement of free phenytoin is warranted. Due to the unavailability of free phenytoin assays in many chemistry analyzers or limitations of immunoassays, chromatographic methods such as liquid chromatography-tandem mass spectrometry (LC-MS-MS) are preferred for the assay of free phenytoin. The sample preparation involved ultrafiltration of serum or plasma to separate free phenytoin. Acetonitrile containing internal standard, phenytoin-d10, was added to the ultrafiltrate. The samples were centrifuged, and supernatants were injected into an LC-MS-MS involving reverse phase Ultra BiPh 5-μm × 50 × 2.1-mm analytical column, and mobile phases, water and methanol containing 0.1% formic acid. The mass/charge (m/z) transitions were as follows: phenytoin -253.0 > 182.2 and 253.0 > 104.00; phenytoin-d10 -263.2 > 192.12. Linearity of the method ranged from 0.1 to 4.0 μg/mL. Within-run and between-run imprecision values were <5% and <10%, respectively. The samples were stable for 2 weeks at 4°C and 4 weeks at -20°C. The method compared well with the laborious liquid-liquid extraction method and did not show any significant ion suppression or enhancement. A simple LC-MS-MS method was developed for the assay of free phenytoin. The method does not require laborious liquid-liquid or solid-phase extraction. The method has high analytical sensitivity, low imprecision, and a wide analytical measurement range.