Stability of Warfarin in Spiked-Saliva Using the Fluorometric HPLC Method

Abstract

Warfarin is an anticoagulant with a narrow therapeutic index ranging from 1-10 µg/mL as well as the ability to distribute into saliva. Therefore, saliva can be selected as an alternative biological fluid in warfarin bioanalysis of therapeutic drug monitoring (TDM) since it is easier and more acceptable, particularly among pediatric and geriatric patients. Stability is an important part of the bioanalysis of warfarin in TDM services. This study aims to conduct a stability of warfarin in spiked-saliva using Fluorometric HPLC at an excitation wavelength of 310 nm and 390 nm emission. Analytes were separated using phosphate buffer:methanol as the mobile phase with a flow rate of 1.0 mL/min and an injection volume of 20µL as well as 150mmx4.5mm C18 as the stationary phase. The standard curve of warfarin with a concentration range of 0-20 ng/mL resulted in a correlation coefficient of 0.999. This study showed that the warfarin stock solution was stable at both 25°C and 4°C for 24 hours and 21 days, respectively. Meanwhile, warfarin in the saliva matrix also remained stable at 25°C for 24 hours and in a storage condition of -20°C for 21 days. In this research, the sample of saliva from patients administered with warfarin that has been treated with a maximum freeze-thaw cycle of 3-fold or 24 hours after preparation could consistently provide accurate data to be used as an approach to making a decision on dosage adjustment and diagnosis of warfarin toxicity in the clinical setting.