Development and Validation of a HPLC‐MS/MS Method for the Determination of Compound 13b in Rat Plasma and Its Application to a Pharmacokinetic Study

Compound 13b, a newly identified anthraquinone derivative, has demonstrated potent efficacy against the Zika virus. To explore the bioavailability and pharmacokinetic properties of compound 13b, a robust and sensitive HPLC-MS/MS method was established and verified to determine its plasma concentration in rats. Sample preparation involved protein precipitation using acetonitrile with testosterone as an internal standard. A Phenomenex Kinetex XB-C18 column (2.1 × 100 mm, 2.6 μm) was utilized for sample separation with gradient elution. The mobile phase, consisting of water and acetonitrile, was dispensed at a flow rate of 1 mL/min. The multiple reaction monitoring mode (MRM) approach was used to detect compound 13b and testosterone, characterized by their respective ionization transitions: m/z 410.2 → 91.1 and m/z 289.2 → 109.2. The method demonstrated superior linearity within rat plasma samples, ranging from 2 to 400 ng/mL, and met all FDA guidelines for bioanalytical method validation. The pharmacokinetic properties were calculated by non-compartmental approach following administration of compound 13b at 14 mg/kg in rats, and the absolute oral bioavailability was found to be 15.45%. Hence, a highly sensitive and rapid HPLC-MS/MS method for measuring plasma concentration of compound 13b in rats has been successfully developed, rigorously validated, and subsequently utilized in a bioavailability and pharmacokinetic study.