Development and Validation of a Simple, Sensitive Fluorescent Method for Eurycomanone Quantification in Tongkat Ali Using Nitrogen-Doped Carbon Quantum Dots and Box–Behnken Design Optimization

Eurycoma longifolia Jack, commonly known as Tongkat Ali, is a medicinal herb traditionally valued for its aphrodisiac properties, with eurycomanone being its principal bioactive quassinoid. This study introduces a novel, cost-effective method for the sensitive detection of eurycomanone using nitrogen-doped carbon quantum dots (N-CQDs) as a fluorescent probe. The spectral characteristics of the probe were carefully analyzed using UV–vis and fluorescence spectrophotometric techniques, and the sensing mechanism was investigated through Stern–Volmer and thermodynamic studies, revealing a static quenching interaction. A Box–Behnken design of experiment was applied to optimize the sensing conditions including pH, incubation time, and probe concentration, ensuring high sensitivity and robustness. A reduced quadratic model was found significant (p value > 0.0001) with pH and N-CQDs concentration being the most influential factors. The method was rigorously validated following ICH guidelines displaying excellent linearity in the range of 0.25–6.0 μg/mL, high sensitivity (LOD 0.067 μg/mL), and high accuracy and precision (RSD < 1.5%). The proposed technique was successfully applied to the analysis of real Tongkat Ali samples, demonstrating its practical utility in herbal analysis. The results showed good agreement with reported chromatographic methods, positioning the technique as a promising alternative to conventional analytical methods.