Rapid, Sensitive and Simple LC-MS/MS Method Development and Validation for Estimation of Phenytoin in Human Plasma

Rapid, sensitive and simple LC-MS/MS method was developed and validated for the determination of phenytoin in human plasma with carbamazepine as internal standard. Gradient elution of phenytoin and carbamazepine were achieved at about 3.5 min with total run time 6 min using Intrada-WP RP, C18, 150 × 3 mm, 3 µ column. The analyte and internal standard were eluted with the gradient flow profile of B: 2 to 45 % (0 to 1 min), 45 to 80 % (1 to 4 min), 80 to 2 % (4 to 5 min). The flow rate was linear from 0 to 5 mL/min up to 5 min and stop at 6 min and the column oven temperature was 50 ± 5 °C. Selectivity and sensitivity of the developed method was improved by selecting electron spray ionization technique in +ve ion mode. The standard plots were observed that the linear within the concentration range of 20.176-15016.936 ng/mL with correlation coefficient ≥ 0.9976. All the validation parameters were within the acceptable limits.