Determination of Hecogenin in Mouse Blood by UPLC–MS/MS and Applied to a Pharmacokinetic Study

ABSTRACT Hecogenin, a steroidal sapogenin from agave plants, exhibits anti‐inflammatory, anti‐tumor, gastroprotective, and diuretic activities. A sensitive ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for quantifying hecogenin in mouse whole blood using swertisin as internal standard. Protein precipitation with acetonitrile enabled simple pretreatment of only 20‐μL blood samples. Separation was achieved on a BEH C18 column with gradient elution, and detection was performed via multiple reaction monitoring in positive electrospray ionization mode. The assay was linear over 1–2000 ng/mL ( r  = 0.9988), with a lower limit of quantification of 1 ng/mL. Intra‐ and interday precision (RSD) were below 11% and 12%, respectively, and accuracy ranged from 93.2% to 110.4%. Matrix effects were negligible, and extraction recovery was 87.5%–91.2%. The validated method was applied to a pharmacokinetic study in male ICR mice following intravenous (5 mg/kg) and oral (10, 20 mg/kg) administration. Noncompartmental analysis revealed low oral absolute bioavailability (10.8%–12.4%).