Abstract
A rapid and simple high pressure liquid chromatography method with mass spectrometry detection was developed and validated for the determination of phenytoin in human plasma. Metaxalone was used as internal standard. The sample preparation involves a rapid and simple procedure based on liquid-liquid extraction. Analysis was performed in less than 3.0 minutes in isocratic mode on a reversed phase C18column (5μ; 50 × 4.6 mm) using a mobile phase composed of acetonitrile-buffer 2 mM ammonium acetate (80:20v/v), pH of buffer adjusted to 3.4 using formic acid, at 0.4 mL min-1 flow rate. The calibration curves were linear in the measured range between 101.2 ng mL-1and 5060.0 ng mL-1. The validated lowest limit of quantification was 101.2 ng mL-1for phenytoin. The mean relative recovery for drug and Internal standard was found to be 78.33% and 77.04%, respectively. The described method has the advantage of being rapid and easy and it could be applied in therapeutic monitoring of these drugs in human plasma.
Highlights
Phenytoin has the molecular formula C15H12N2O2 and the chemical name 5,5-diphenylimidazolidine-2,4-dione with molecular weight of 252.268 g mol-1
Calibration graphs were constructed by plotting peak-area ratios of phenytoin to internal standard against nominal concentration (101.2, 253.0, 506.0, 759.0, 1,012.0, 2,530.0, 4,08.0 and 5,060.0 ng mL–1)
Extraction recoveries of phenytoin from spiked samples were determined by comparing the peak areas obtained by extraction of freshly prepared plasma extracts at low, medium and high concentration levels, with those found by direct injection of an aqueous standard solution[18] at equivalent concentration (n= 6)
Summary
Phenytoin has the molecular formula C15H12N2O2 and the chemical name 5,5-diphenylimidazolidine-2,4-dione with molecular weight of 252.268 g mol-1. By promoting sodium efflux from neurons, phenytoin tends to stabilize the threshold against hyper excitability caused by excessive stimulation or environmental changes capable of reducing membrane sodium gradient This includes the reduction of posttetanic potentiation at synapses. Series of working standard solutions with concentrations of 2024.0, 5060.0, 10120.0, 15180.0,20240.0, 50600.0, 80960.0 and 101200.0 ng mL-1 were prepared by dilution of aliquots of stock with methanol and water in the proportion of 60: 40 v/v. To prepare calibration standards and quality control samples, 25 μL of the various diluted working calibration standard and 50 μL of internal standard (10.04 μg mL-1) solution were added to blank plasma to a final volume of 0.50 mL; the contents of the tube vortexes for 30 sec. Dissolved the residue with 500 μL of mobile phase and injected into the LC-MS/MS system
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