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

Document Type

Article

Source of Publication

Methods in Molecular Biology

Publication Date

1-1-2026

Abstract

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.

ISSN

1064-3745

Publisher

Springer US

Volume

3026

First Page

69

Last Page

77

Disciplines

Life Sciences

Keywords

Active form, Cytokinins, Non-plant systems, Plant hormones, Signalling molecules, Storage form, Two-component systems

Scopus ID

105034818379

Indexed in Scopus

yes

Open Access

no

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