Improved HPLC method for the determination of ribavirin concentration in red blood cells and its application in patients with COVID-19

Ribavirin is a synthetic, broad-spectrum antiviral drug. Ribavirin is recommended as an antiviral drug in the Interim Guidance for Diagnosis and Treatment (the seventh edition) of COVID-19. The ribavirin levels in red blood cells may be closely related to both its efficacy and adverse drug reactions. In this study, a simple and fast HPLC-UV method was established to determine the concentrations of total ribavirin in the red blood cells of 13 patients with COVID-19. Phosphorylated ribavirin was dephosphorylated by phosphatase incubation to obtain the total amount of ribavirin in red blood cells. The chromatographic column was an Atlantis C₁₈ . The recoveries were 85.45-89.05% at three levels. A good linear response was from 1 to 200 μg/ml, with a correlation coefficient of r²  = 0.9991. The concentration of total ribavirin in the red blood cells of the patients ranged from 30.83 to 133.34 μg/ml. The same samples without phosphatase incubation ranged from 4.07 to 20.84 μg/ml. About 85% of ribavirin was phosphorylated in red blood cells. In addition, we observed changes in these patients' hematological parameters and found that the erythrocyte, hemoglobin and hematocrit declined to the lowest levels on the fifth day after discontinuation of ribavirin (p < 0.05).