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

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%).