Cytokinin Quantification in Plants

Bieleski buffer (60% methanol, 25% CHCl₃, 10% HCOOH and 5% H₂O) was used as extraction solvent (50 μl per sample). Tritium-labelled (10⁵ dpm) or unlabelled (1 pmol) cytokinin standards were added to the sample extracts during the method optimization to determine the recoveries of the StageTip purification procedure. To validate the quantification of the endogenous cytokinin levels in A. thaliana seedlings, roots and shoots, the following stable isotope-labelled cytokinin internal standards (IS) were added as internal tracers at a concentration 0.5 pmol of each compound per 50 μl of Bieleski buffer: [¹³C₅]cZ, [¹³C₅]tZ, [²H₅]tZR, [²H₅]tZ7G, [²H₅]tZ9G, [²H₅]tZOG, [²H₅]tZROG, [²H₅]tZMP, [²H₃]DHZ, [²H₃]DHZR, [²H₃]DHZ9G, [²H₇]DHZOG, [²H₃]DHZMP, [²H₆]iP, [²H₆]iPR, [²H₆]iP7G, [²H₆]iP9G, [²H₆]iPMP. The plant material was placed in 2.0 ml microcentrifuge tubes and extracted in Bieleski solvent using a MM 301 vibration mill (Retsch GmbH & Co. KG, Haan, Germany) at a frequency of 27 Hz for 3 min after adding 3 mm tungsten carbide beads (Retsch GmbH & Co. KG, Haan, Germany) to increase the extraction efficiency. The tube content was ultrasonicated for 3 min and then stirred for 30 min at 4°C. After centrifugation (10 min, 15,000 rpm, 4°C) the supernatants (50 μl aliquots) were immediately transferred onto StageTips and purified according to the following protocol.
The PT-SPE was performed in self-packed StageTips by placing a very small disk of matrix in an ordinary pipette tip. Commercially available matrix of poly-tetrafluoroethylene containing reversed-phase octadecyl-bonded silica phase (C18) or poly(styrene-divinylbenzene) (SDB) copolymer modified with sulfonic acid groups to make it more hydrophilic (SDB-RPS Disk) was normally used. Alternatively, ion-exchange sorbent including sulfonic acid as cation exchanger (Cation-SR Disk) was also employed. The procedure shown in Additional file 5 was described by Rappsilber et al. [23 (link),26 (link)]. Small disks (approximately 1.0 mm diameter, 0.5 mm thickness) were cut out manually from the Empore^TM High Performance Extraction Disk placed on a clean surface (Petri dish) using a hollow tool cutter (blunt-ended syringe needle). The cutter was gently pressed into the Empore disk and the material penetrated to the inside of the needle. Subsequently, the cutter was placed inside a pipette tip (disposable GELoader® Tip, 100 μl, from Eppendorf). The small disk was then released using a plunger (rod) that fitted into the needle (both parts from Hamilton) and pressed gently repeatedly into place using the weight of the plunger. After removing the cutter and plunger, the single-StageTip was finished. Additional disks were added the same way to produce combined multi-StageTips.
The Empore sorbents were tested individually (C18, SDB-RPS, and Cation-SR) or in combination (C18/SDB-RPS, C18/Cation-SR, C18/SDB-RPS/Cation-SR). All solutions were loaded from the top of the tip in a volume of 50–100 μl using a pipette. The prepared StageTip was inserted into a hole at the centre of the lid of the microcentrifuge tube (1.5 ml) and placed in a centrifuge after solvent pipetting (Figure 2). Prior to loading the sample the StageTip sorbents were activated with 50 μl acetone (by centrifugation at 1,500 rpm, 15 min, 4°C), 50 μl water (1,500 rpm, 15 min, 4°C), 50 μl methanol (1,500 rpm, 15 min, 4°C), 50 μl water (1,500 rpm, 15 min, 4°C), equilibrated with 50 μl 50% (v/v) nitric acid (1,000 rpm, 20 min, 4°C), 50 μl water (1,500 rpm, 15 min, 4°C) and 50 μl Bieleski solvent (1,500 rpm, 15 min, 4°C). Afterwards, the samples were loaded in extraction buffer (500 rpm, 45 min, 4°C). The tips were washed using 50 μl methanol (1,500 rpm, 15 min, 4°C) and elution of samples was performed with 50 μl of 0.5 M NH₄OH in 60% MeOH (500 rpm, 45 min, 4°C). Eluates were collected into new clean microcentrifuge tubes and directly mixed with scintillation buffer prior to measurement of radioactivity or evaporated to dryness in a Speed-Vac concentrator RC1010 (Jouan, Winchester, UK) and dissolved in 20 μl of mobile phase prior to UHPLC-MS/MS analyses.