Towards the identification of alkaline phosphatase binding ligands in Li-Dan-Hua-Shi pills: A Box-Behnken design optimized affinity selection approach tandem with UHPLC-Q-TOF/MS analysis

•Magnetic alkaline phosphatase linked microspheres were fabricated and characterized.•Excellent magnetic behavior of microspheres resulting to its easy collection.•Magnetic affinity selection approach for fishing alkaline phosphatase binding ligands in herbal mixtures. Alkaline phosphatase conjugated magnetic microspheres were synthesized via amide reaction, and employed as an effective adsorbent in affinity selection of binding ligands followed by UHPLC-Q-TOF/MS analysis. The analytical validity of the developed approach was evaluated under optimized conditions and the following figures of merit were obtained: linearity, 0.01–0.5 g L−1 with good determination coefficients (R2 = 0.9992); limits of detection (LODs), 0.003 g L−1; and limits of quantitation (LOQ), 0.01 g L−1. The precision (RSD%) of the proposed affinity selection approach was studied based on intra-day (0.8%) and inter-day (1.3%) precisions. Finally, the adsorbent was successfully applied to identification of binding ligands in Li-Dan-Hua-Shi pills and good recoveries were obtained in the range from 96.9 to 99.4% (RSDs 1.6–3.0%).