Validated HPLC Analysis of Puerarin in Pueraria lobata–Derived Exosome‐Like Nanoparticles With Intestinal Barrier Enhancement

ABSTRACT A high‐performance liquid chromatography with diode array detection (HPLC‐DAD) method was developed and validated to quantify puerarin encapsulated in exosome‐like nanoparticles derived from Pueraria lobata (ENsP). The method demonstrated high specificity, excellent linearity ( r 2 = 0.9999), and low limits of detection (LOD: 0.60 µg/mL) and quantification (LOQ: 1.83 µg/mL). Matrix effects were negligible, and accuracy and precision tests revealed high recovery rates (95.8%–105.5%) with low relative standard deviations (0.58%–4.10%). Horwitz ratio values (0.15–0.66) and relative expanded uncertainties (13.8%–16.6%) complied with CODEX standards (≤22%). The puerarin content in ENsP was determined to be 1.08 ± 0.02 mg/g, and its stability was maintained over 10 days at 4°C. In a cyclophosphamide (CPA)‐induced immunosuppressed BALB/c mouse model, oral administration of ENsP significantly improved small intestinal morphology, including restoration of intestinal length and the villus‐to‐crypt ratio, and enhanced expression of barrier‐related genes and proteins ZO‐1 and MUC2. These results highlight ENsP as a stable and biocompatible carrier with functional bioactivity, offering promising potential for pharmaceutical and nutraceutical applications targeting intestinal barrier integrity. Practical Applications This study developed and validated an HPLC‐DAD method for the quantification of functional compounds in exosome‐like nanoparticles derived from Pueraria lobata (ENsP). This advancement establishes a foundation for quality control and the further development of functional food ingredients.