Modification of a high-performance liquid chromatography method for quantitative determination of rivaroxaban concentration in blood

Introduction. Achieving therapeutic concentrations of direct oral anticoagulants (DOAC) and, accordingly, personalization of anticoagulant therapy in certain groups of patients are becoming increasingly important. To optimize drug monitoring in routine clinical practice, it is necessary to develop and test sensitive and selective methods that can quantitatively determine the content of DOAC in the blood, including rivaroxaban.

Aim: To modify the method for determining the concentration of rivaroxaban in the blood using high-performance liquid chromatography (HPLC), as well as testing the selected conditions for determining the concentration of the drug in the whole venous blood of patients.

Material and Methods. For the first stage of the work, the samples for the study were blood samples (n = 20) from healthy volunteers (n = 5) not taking medications. Blood was collected from a peripheral vein using a vacuum system in a volume of 6 ml. Blood collection tubes with four different fillings were used. Before preparation, the samples were divided into 6 parts and each was supplemented with a solution of rivaroxaban of different concentrations, prepared from the pure substance Rivaroxaban (India). Next, liquid extraction of rivaroxaban with acetonitrile from a sample of whole venous blood was carried out with parallel elimination of the interfering effect of proteins by precipitation with nickel chloride. The separation of the sample components was performed by reversed-phase high-performance liquid chromatography on a liquid chromatograph (1260 Infinity II LC). The analytical signal on a diode-array detector (1260 DAD WR, Agilent Technologies) was recorded at two wavelengths of 254 nm and 280 nm. For the second stage of the work, the samples for the study were whole venous blood samples from patients taking rivaroxaban (n = 54). Blood is collected in the morning, on an empty stomach, 12 hours after taking the last dose of the drug.

Results. The authors investigated and established the factors influencing the decrease in the detection limit of rivaroxaban (Bayer, Germany) in the blood when determining it by HPLC using an Agilent 1260 liquid chromatograph with a diode array detector (Agilent Technologies, Germany). To assess the degree of rivaroxaban extraction and select the working wavelength, calibration dependences of the anticoagulant in the initial acetonitrile solutions and graphs of changes in the peak areas of rivaroxaban from its content in the blood were constructed: at a wavelength of 254 nm (R2 = 0.98) and 280 nm (R2 = 0.99). The dependence function at a wavelength of 254 nm has a larger slope, which makes it possible to reduce the detection limit of the substance being determined. The silicon dioxide content in the tube does not significantly affect the analytical signal of rivaroxaban (minimum detection limits 0.25 µg/ml, minimum determination limits 0.77 µg/ ml), and does not introduce additional error into the accuracy of the method. The median concentration of rivaroxaban was 0.32 µg/ ml (0.26; 0.49) 12 hours after taking the drug.

Conclusion. The use of a modified HPLC method with a wavelength of 254 nm selected for detecting the analytical signal makes it possible to reduce the detection limit of the substance being determined and increase the range of concentrations being determined. And the use of tubes with silicon dioxide can be recommended for decreasing the influence of the matrix effect on the quantitative determination of rivaroxaban in blood and increasing the accuracy of the method.

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