A simple HPLC method for the quantification of mizoribine in human serum: Pharmacokinetic applications

A simple high-performance liquid chromatographic (HPLC) method was developed and validated for the quantification of mizoribine in human serum. After the addition of 70% perchloric acid and 3-methylxanthine (50 μg/mL, internal standard) to human serum, the samples were mixed and centrifuged at 12,000 rpm (1432g) for 10 min. The supernatant was injected onto a C₁₈ column eluted with a mobile phase of 20 mM Na₂HPO₄ and methanol (93:7, v/v, pH 3) containing 0.04% octanesulfonic acid and detected utilizing an ultraviolet detector at 275 nm. The linear calibration curve was obtained in the concentration range of 0.1-4.0 μg/mL and the lower limit of quantification was 0.1 μg/mL. This method was validated with selectivity, linearity, precision and accuracy. In addition, the method was successfully applied to estimate the pharmacokinetic parameters of mizoribine in Korean subjects following an oral administration of 100 mg mizoribine (two Bredinine® 50 mg tablets). The maximum serum concentration (C_max) of 2.30 ± 0.83 μg/mL was reached 2.27 ± 0.66 h after an oral dose. The mean AUC_0-12h and the elimination half-life (t_1/2) were 13.2 ± 4.79 μg h/mL and 3.10 ± 0.74 h, respectively.