Determination of free and total levels of phenytoin in human plasma from patients with epilepsy by MEKC: An adequate alternative to HPLC

Abstract

Phenytoin is a widely used anti-seizure agent, and a good correlation is observed between its concentration in plasma and the clinical effect. We developed a selective CE with UV detection at 200 nm for analysis of free and total levels of phenytoin in human plasma based on MEKC. A sample pretreatment by liquid-liquid extraction with ethyl acetate for determination of total level of phenytoin and serum ultrafiltrate was prepared by ultrafiltration technique (ultrafiltration membrane 30 kDa; 2000 g, 10 min) for determination of free level of phenytoin and subsequent quantification by MEKC was used. MEKC was performed in Tris buffer (25 mM; pH 10.5) containing SDS (180 mM) and EG (13%) as BGE. Hydrodynamic injection for phenytoin determination (0.5 psi 5 s for total level, 2 psi 5 s for free level) was used to introduce samples. The separation voltage was set at 20 kV. Data obtained by MEKC were compared with the results by a validated HPLC method. The MEKC assay of phenytoin exhibited a very good correlation with respect to HPLC by Bland-Altman method. The equations for the Passing-Bablok regression line were as follows: for total level: MEKC=1.0143 x HPLC+0.0976, r(2)=1; for free level: MEKC=1.0013 x HPLC-0.0016, r(2)=1. The proposed method was applied successfully to monitor free and total levels of phentoin in 20 patients with seizures.

Determination of phenytoin in human plasma by a validated hplc method: application to therapeutic drug monitoring study

“…PHT, following the oral ingestion, precipitates in stomach. A good correlation is showed between its plasma concentration and clinical effect 6 . Although peak plasma levels occur 3-12 hours after a single dose, its absorption can be extended up to 2 weeks, especially at large overdose.…”

“…HPLC coupled with ultraviolet (UV) detection [12][13][14][15][16][17][18] , flourescence detection 19 , tandem mass spectrometry (MS-MS) [20][21][22][23] ; gas chromatography (GC) coupled with mass spectrometry (MS) 18,24 and capillary electrophoresis (CE) coupled with UV 6 are the reported methods that were used for determination of PTH in biological samples. In addition to that, colouremetric based determination methods 25 , radioimmunoassay 18 , enzyme immunoassay (EMIT) 18,26 , fluorescence polarization immunoassay (FPIA) 27 and spectrophotometric assay 18 have been often used monitorization of PHT.…”

Antiepileptic Effects of Lacosamide Loaded Polymers Implanted Subdurally in GAERS

“…For each sample, the release medium solution was collected and replenished with fresh aCSF at various time points and stored at −20 ∘ C prior to HPLC analysis being undertaken. The eluted samples were analyzed by HPLC using a modified method on an Agilent 1260 Infinity HPLC system [19]. An Atlantis Ⓡ T3 C18 column (250 mm × 4.6 mm, 5 m) was employed as the analytic column and set at 40 ∘ C. The mobile phase was composed of water, acetonitrile, and methanol (65 : 26.2 : 8.8, v/v/v), and the injection volume was 10 L with the flow rate of 0.8 mL/min.…”

PETINIA Total Phenytoin Assay on the Vista 1500 Analyzer Is Not Suitable for Monitoring Free Phenytoin Concentration if Ultrafiltration Is Performed at Room Temperature

“…Kilpatrick et al 2 reported that unbound phenytoin concentration reflected the clinical status of a patient better than the total phenytoin concentration. 10,11,12,13 However, phenytoin protein binding is temperature dependent, and if ultrafiltration is performed at room temperature (258C), it does not reflect in vivo free phenytoin concentration because the physiological temperature is 378C. The authors recommended monitoring of free phenytoin in trauma patients.…”