Green profiling of lotus alkaloids using DES-based micro-matrix solid-phase dispersion: Tissue distribution and bioactivity-linked quality evaluation

Lotus alkaloids are bioactive constituents of Nelumbo nucifera, but their determination in plant matrices remains challenging. This study developed a method for the determination of six lotus alkaloids and evaluated their tissue distribution, quality, and acetylcholinesterase-associated activity. A micro-matrix solid-phase dispersion procedure was established using a deep eutectic solvent-functionalized ZSM-5 molecular sieve, followed by high-performance liquid chromatography-tandem mass spectrometry. The method was optimized and validated for selectivity, linearity, sensitivity, precision, accuracy, matrix effect, stability, and robustness, and was applied to 67 batches of lotus plumules, seed kernels, and leaves. Multivariate analysis and an acetylcholinesterase inhibition assay were further employed for quality evaluation and spectrum-effect analysis. The six alkaloids showed good linearity (R² > 0.992), with limits of detection and quantification of 0.0001-0.0054 and 0.0006-0.0215 μg/mL, respectively. Precision and repeatability were below 3.27% and 4.14%, respectively, while recoveries ranged from 83.13% to 113.29%. The method showed acceptable matrix effects and robustness under deliberate chromatographic variations. Alkaloid accumulation was strongly tissue-dependent, with plumules enriched in bisbenzylisoquinolines and leaves characterized by higher aporphine alkaloids. Marked quality differences were observed between pharmacy- and online-purchased leaf samples. Lotusine, liensinine, isoliensinine, and neferine were identified as candidate quality markers associated with acetylcholinesterase inhibition. The proposed method provides a sensitive, reliable, and miniaturized approach for the analysis of structurally diverse lotus alkaloids, while integrating chemical profiling with bioactivity analysis to provide an efficacy-oriented strategy for quality evaluation of lotus-derived materials.