Rapid, Sensitive and Simple LC-MS/MS Method Development and Validation for Estimation of Phenytoin in Human Plasma by using Deuterated Internal Standard

Abstract

A rapid, sensitive, accurate and precise high-performance liquid chromatography–tandem mass spectrometric (HPLC– MS/MS) method equipped with Electro Spray Ionization (ESI) source, operating in the Positive ion and Multiple Reaction Monitoring (MRM) mode was developed and validated for the estimation of Phenytoin in human plasma using Phenytoin D10 as an internal standard. Liquid-liquid extraction (LLE) technique was used to extract analyte and ISTDs from 0.2mL human plasma. The analytical separation was carried out in a reverse phase liquid chromatography by using C18 (150 x 4.6mm, 5µm) column, and 2mM Ammonium Acetate in water (pH 6.3): Methanol (30:70% v/v) mobile phase at 1mL/min in isocratic mode. Analytes were monitored in multiple reactions monitoring (MRM) mode using the respective [M+H]⁺ ions, m/z 253.10→ 182.30 for Phenytoin and m/z 263.30 → 192.20 for the internal standard, respectively. The Phenytoin and Phenytoin D10 retained at about 2.50 and 2.46 minutes respectively with total run time of 4 minute. The response of the LC-ESI-MS/MS method for both analyte and ISTD was linear over the dynamic range of 60-12000ng/mL with correlation coefficient r2 ≥ 0.9963. The Accuracy was well within the accepted limit of ± 20% at lower limit of quantification (LLOQ) and ± 15% at all the other concentrations in the linear range. Recovery of drug and ISTD was 87.52% and 86.42%, respectively. This method was fully validated for all the validation parameters and Stability studies. After optimization, the Bioanalytical method was validated according to USFDA, EMA and ANVISA guidelines for Bioanalysis.