Optimizing Cytokinin Purification Using StageTips

To test the selectivity, affinity, and capacity of different StageTip sorbent combinations, samples containing ³H-labelled CK standards ([³H]cZ, [³H]tZR, [³H]iPR) were used. Firstly, to characterize the potential sorbents (C18, SDB-RPS, and Cation-SR), the single-StageTip experiments were performed by pipetting 50 μl of the selected ³H-CK standard (10⁵ dpm) in Bieleski buffer directly onto a PT-SPE column. Secondly, different combinations of sorbents (multi-StageTips) were tested using the same spiked Bieleski buffer with and without plant matrices (1.0 and 2.0 mg FW) to monitor the loading capacity and extraction recovery. For all samples, the yields of ³H-CKs after passage through StageTips filled with different sorbents and their combinations were determined. Thirdly, non-spiked and spiked (with 0.1, 1 and 10 pmol of unlabelled CK standards) samples of 10-day old Arabidopsis thaliana seedlings (1–5 mg FW) were used to verify the reproducibility, sensitivity and accuracy of the method. Aliquots of the extracts were processed using the developed purification procedure (Figure 2), then analyzed by UHPLC-ESI(+)-MS/MS. The intraday reproducibility was evaluated by repeating the process for three times within one day, and the interday reproducibility was investigated on three successive days. The recovery of added authentic cytokinin standards were then determined from each series of extracts, based on the amounts of endogenous compounds, calculated from non-spiked samples, which subsequently served as reference levels.