Determination of free and total levels of phenytoin in human plasma from patients with epilepsy by MEKC: an adequate alternative to HPLC.
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.
Lin
Ping-Chih
PC
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.
Hsieh
Ya-Hui
YH
Liao
Fen-Fen
FF
Chen
Su-Hwei
SH
D000328:Adult; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004827:Epilepsy / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D010672:Phenytoin / Q000097:blood* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D014325:Tromethamine; D055815:Young Adult
D014325:Tromethamine; D012967:Sodium Dodecyl Sulfate; D010672:Phenytoin