A simple sample preparation approach based on hydrophilic solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry for determination of endogenous cytokinins

• A simple and efficient method was developed for the determination endogenous CKs.
• CKs were purified by one-step HILIC SPE based on hydrophilic interaction.
• Endogenous CKs can be quantified from only 20 mg plant sample.

Cytokinins (CKs), a vital family of phytohormones, play important roles in the regulation of shoot and root development. However, the quantification of CKs in plant samples is frequently affected by the complex plant matrix. In the current study, we developed a simple, rapid and efficient hydrophilic interaction chromatography-solid phase extraction (HILIC–SPE) method for CKs purification. CKs were extracted by acetonitrile (ACN) followed by HILIC–SPE (silica as sorbents) purification. The extraction solution of plant samples could be directly applied to HILIC–SPE without solvent evaporation step, which simplified the analysis process. Moreover, with HILIC chromatographic retention mechanism, the hydrophobic co-extracted impurities were efficiently removed. Subsequently, CKs were separated by RPLC, orthogonal to the HILIC pretreatment process, and detected by tandem mass spectrometry. The method exhibits high specificity and recovery yield (>77.0%). Good linearities were obtained for all eight CKs ranging from 0.002 to 100 ng mL−1 with correlation coefficients (r) higher than 0.9927. The limits of detection (LODs, signal/noise = 5) for the CKs were between 1.0 and 12.4 pg mL−1. Reproducibility of the method was evaluated by intra-day and inter-day measurements and the results showed that relative standard deviations (RSDs) were less than 10.5%. Employing this method, we successfully quantified six CKs in 20 mg Oryza sativa leaves and the method was also successfully applied to Brassica napus (flower and leaves).