Validation of HPLC-UV method for the phenytoin determination in spiked-saliva for TDM application

Phenytoin (PHT) is an antiepileptic drug with nonlinear pharmacokinetics, highly-protein bound and is known to have a narrow therapeutic range, making it requires a therapeutic drug monitoring (TDM) through an examination of drug levels in blood. In addition to the limited availability of validated bioanalytical methods, the use of blood samples becomes one of the factors that complicate TDM application of antiepileptic drugs, especially in pediatric and geriatric patients. Instead of blood, saliva as an alternative biological fluid can be used not only due to the non-invasive sampling technique associated with it but also because the salivary PHT concentrations are significantly correlated with free concentrations in the blood that provide pharmacological effects. This study aimed to validate the method for determination of PHT levels in spiked-saliva on the basis of FDA criteria. The optimum conditions were obtained using a methanol mobile phase of aqua pro-injection (55:45) with a stationary phase of C8 (4.6 × 150 mm; 5 µm) from SunFire™ and SM7 injector with a UV detector at 200nm wavelength. The standard curve of PHT in spiked-saliva with a concentration range of 0.3 - 5 µg/mL showed a correlation coefficient value of 0.999. The LoD (Limit of Detection) reached 0.167 µg/mL. The results of selectivity, accuracy, precision, and recovery tests have met the validation parameter criteria established by FDA.