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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.

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Extraction and Analysis of Cytokinins from Mammalian Tissues by HPLC–HRMS

  • 实验方案

  • 发表日期：

    2026年

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  • Muhammad Naseem,
  • Kenesha Wilson,
  • Feras Lafi,
  • Elena Bencurova,
  • Khalid Muhammad,
  • Thomas Dandekar,
  • Alfonso Albacete,
  • Eman M. Othman

摘要

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.