Evaluation of Antiepileptic Drugs' Stability in Oral Fluid Samples.

Epilepsy affects approximately 50 million people worldwide, with antiepileptic drugs (AEDs) remaining the cornerstone of treatment. Due to their narrow therapeutic windows, AEDs are ideal candidates for therapeutic drug monitoring (TDM). Oral fluid is increasingly considered a viable alternative to blood and urine, as it reflects the free (active) concentration of many AEDs. Its non-invasive collection, which does not require trained personnel, makes it particularly suitable for TDM in paediatric and geriatric populations. However, as samples are often stored for extended periods before analysis, analyte stability becomes a critical concern. This study aimed to evaluate the stability of four commonly used AEDs in dried saliva spot (DSS) samples. Phenobarbital, phenytoin, carbamazepine, and carbamazepine-10,11-epoxide were analysed in oral fluid samples collected via spitting and stored as DSSs. Quantification was performed using high-performance liquid chromatography with diode array detection (HPLC-DAD). Design of experiments tools were used to assess the effects of preservatives, storage temperatures, light exposure, and storage durations on analyte stability. Optimal conditions were refrigeration in the dark, with a low concentration of ascorbic acid as preservative. Samples at 10 µg/mL remained stable for 14 days longer than those without preservative or reported in previous studies. Unexpectedly, at 0.5 µg/mL, analytes in samples without preservative showed greater stability. To our knowledge, this is the first study combining DSS and HPLC-DAD to assess the stability of these AEDs in oral fluid, providing valuable insights for non-invasive TDM strategies and supporting the feasibility of saliva-based monitoring in clinical settings.