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. 2011;6(12):e28182.
doi: 10.1371/journal.pone.0028182. Epub 2011 Dec 2.

Measurement of warfarin in the oral fluid of patients undergoing anticoagulant oral therapy

Silvia Ghimenti  1 , Tommaso Lomonaco, Massimo Onor, Laura Murgia, Aldo Paolicchi, Roger Fuoco, Lucia Ruocco, Giovanni Pellegrini, Maria Giovanna Trivella, Fabio Di Francesco
Affiliations

Measurement of warfarin in the oral fluid of patients undergoing anticoagulant oral therapy

Silvia Ghimenti et al. PLoS One. 2011.

Abstract

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 controls.

Methodology: 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.

Findings: The method was free from interference and matrix effect, linear in the range 0.2-100 ng/mL, with a detection limit of 0.2 ng/mL. Its coefficient of variation was <3% for intra-day measurements and <5% for inter-day measurements. 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: Warfarin diffuses from blood to oral fluid. The method allows to measure its concentration in this matrix and to analyze correlations with INR and other parameters.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HPLC chromatograms of: A) a standard working solution with a warfarin concentration of 1.5 ng/mL; B) a control oral fluid sample (COFS); C) a standard oral fluid sample (SOFS) with a warfarin concentration of 1.4 ng/mL; D) a patient oral fluid sample (POFS) with a warfarin concentration of 1.4 ng/mL.
(Chromatographic conditions: Poroshell 120 EC-C18 column, 65% PBS and 35% methanol mobile phase, flow rate of 0.7 mL/min; injection volume 25 µL).
Figure 2
Figure 2. Principal component analysis of patient data.
A) score plot, B) loadings plot. Legend: pH of the oral fluid (pH), warfarin concentration in the oral fluid (WC), warfarin dose (Dose), INR.
Figure 3
Figure 3. Effect of the warfarin weekly dose on A) INR and B) warfarin concentration in oral fluid.
Figure 4
Figure 4. Correlation between the concentration of warfarin and pH in the oral fluid.
Figure 5
Figure 5. Warfarin concentration in oral fluid versus INR.
Samples with a pH level higher than 7.2 are indicated by full dots: A) all the samples, B) detail of samples with a pH level higher than 7.2.
Figure 6
Figure 6. Correlation between calculated concentration of warfarin in plasma and International Normalized Ratio for patients with a pH≥7.2.
See this image and copyright information in PMC

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