Extraction and Analysis of Cytokinins from Mammalian Tissues by HPLC-HRMS.

Cytokinins (CKs) are adenine-derived plant hormones regulating almost every aspect of plant growth and development. But CK-like compounds may also occur in microbial as well as mammalian systems. Detecting CKs in animal matrices is challenging due to low abundance and complex backgrounds. We describe a high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) protocol for CK extraction, purification, and quantification. Samples are frozen or lyophilized, homogenized, and extracted with methanol: water (80:20, v/v) containing stable isotope labeled CKs as internal standards. Solid-phase extraction (SPE) with C18 cartridges removes proteins and lipids, and purified eluates are dried, reconstituted, and analyzed by reversed-phase HPLC coupled to an Orbitrap mass spectrometer. Accurate-mass detection (≤5 ppm) and diagnostic fragmentation confirm identity, while isotope-dilution calibration enables picomolar sensitivity. This method provides high recovery (>75%) and selectivity, enabling investigation of CK occurrence in ex-planta cellular systems.