Oasis max cartridge column

Three sets of frozen plant tissues (300 mg) were homogenized five times with 1500 rpm for 30 s in 80% (v/v) MeOH containing 5% (v/v) FA as the extraction solution containing one 10 mm stainless bead using Shake Master NEO (Bio Medical Science, Tokyo, Japan). d₂-KA (10 ng) was added to the extraction solution as an internal standard. The homogenized samples were spun (200 rpm) at 4 °C overnight. The homogenates were then centrifuged ( 1570×g, 5 min, 4 °C) and endogenous KA was re-extracted from the resulting pellets in the same solution twice. The combined extracts were evaporated to a water phase using a vacuum evaporator centrifuge (Iwaki Glass, Chiba, Japan) and purified using an Oasis MCX cartridge column (60 mg, 3 cc, Waters) activated with MeOH and pre-equilibrated with 5 mM FA. The evaporated samples were dissolved in 5 mM FA and loaded onto the cartridges, which were washed with 5 mM FA. Columns were then run to dryness, and KA was eluted with MeOH. Elutes were evaporated to dryness. The sample was dissolved in 25 mM NH₄HCO₃ before loading onto an Oasis MAX cartridge column (60 mg, 3 cc, Waters). The column was first activated with MeOH then equilibrated with 25 mM NH₄HCO₃ before loading the sample. Columns were then washed with MeOH and KA was eluted with 0.2 M FA in MeOH, which was evaporated to dryness.