Development of a UPLC‐MS/MS Method for Bioanalysis of Ethoxysanguinarine and Its Application in Pharmacokinetic Study of Ethoxysanguinarine Nanoemulsion

ABSTRACT Ethoxysanguinarine (ETSG), a benzophenanthridine alkaloid, exhibits diverse biological activities, including antibacterial, antifungal, anti‐inflammatory, antioxidant, and anti‐tumor effects. Despite these properties, limited research exists on ETSG in vivo pharmacokinetics due to its poor solubility and low bioavailability. In this study, we developed a rapid and specific UPLC‐MS/MS method for ETSG bioanalysis. Sample preparation involved one‐step protein precipitation using methanol and phellodendrine as an internal standard (IS). The Waters HSS T3 column (2.1 * 50 mm, 1.8 μM) employed a gradient elution with mobile phases A (2 mmol/L ammonium formate aqueous solution‐formic acid [99.8:0.2, v/v]) and B (methanol‐formic acid [99.8:0.2, v/v]). Mass analysis via Waters Q‐mass spectrometer utilized positive scan mode and multiple reaction monitoring. ETSG and IS were detected at m/z 332.0 → 274.0 and 342.0 → 177.0, respectively, within 7.0 min. The method demonstrated excellent precision, accuracy, recovery, and stability, with a linear calibration curve (1.1–560 ng/mL) and strong correlation coefficient (0.9984). Successful pharmacokinetic evaluation in Sprague–Dawley rats included intravenous ETSG administration and intragastric ETSG nanoemulsion/suspension. This method enables steroidal saponin analysis from ETSG in biological samples.