Phenytoin in spiked-saliva for TDM service by medical laboratories: A stability test through HPLC-UV

The clause 5.4.7 in ISO SNI 15189: 2012 requires medical laboratories to have a procedure for the time limit of sample storage, including for therapeutic drug monitoring (TDM) services in the clinical setting. In TDM, saliva is used as a biological matrix due to the non-invasive and convenient collection technique especially for the children and geriatric patients. Phenytoin (PHT) concentration in saliva is known to significantly correlate with its free serum concentration in respect of the clinical response. This research aimed to perform a stability test of PHT in spiked-saliva by utilizing HPLC-UV for the service protocol to monitor PHT concentration in TDM services. PHT concentration was determined using Reversed-Phase HPLC at 200 nm wavelength with C8 (4.6 x 150 mm; 5 µm) stationary phase and methanol: water (55:45) mobile phase. The stability test was conducted on PHT in spiked-saliva at 1.5 and 4 µg/mL concentrations stored at a room temperature and -20°C for one month. The results showed that PHT stock solution in methanol solvent was stable for up to 30 days at 4°C. However, the saliva samples containing PHT were stable only for less than 6 hours at a room temperature or when stored for less than 7 days at -20°C. In contrast, the analyte of extracted PHT in spiked-saliva remained stable for a maximum of 24 hours of storage at room temperature. We recommend that samples of patients’ saliva which are extracted and stored at 4°C remain usable for an adjustment of dose and PHT toxicity diagnosis in clinical settings.