Measurement of Warfarin in the Oral Fluid of Patients Undergoing Anticoagulant Oral Therapy

Background: Patients on warfarin therapy undergo invasive and expensive checks for the coagulability of their blood. No information on coagulation levels is currently available between two checks. Methods: A method was developed to determine warfarin in oral fluid by HPLC and fluorimetric detection. The chromatographic separation was performed at room temperature on a C-18 reversed-phase column, 65% PBS and 35% methanol mobile phase, flow rate 0.7 mL/min, injection volume 25 μL, excitation wavelength 310 nm, emission wavelength 400 nm. Results: Characteristics of the method: no interference and matrix effect, linear calibration curve (range 0,2 – 100 ng/mL), limit of detection = 0,2 ng/mL, within-run repeatability ≤ 6%, total standard deviation = 8% at a warfarin concentration level of 2,9 ng/ml. The average concentration of warfarin in the oral fluid of 50 patients was 2.5 ± 1.6 ng/mL (range 0.8-7.6 ng/mL). Dosage was not correlated to INR (r = -0.03, p = 0.85) but positively correlated to warfarin concentration in the oral fluid (r = 0.39, p = 0.006). The correlation between warfarin concentration and pH in the oral fluid (r = 0.37, p = 0.009) confirmed the importance of pH in regulating the drug transfer from blood. A correlation between warfarin concentration in the oral fluid and INR was only found in samples with pH values ≥ 7,2 (r = 0.84, p = 0.004). Conclusions: The method allows to measure warfarin concentration in oral fluid resulting from the diffusion from blood, and to analyze correlations with INR and other parameters.