Mcx spe cartridge

Chokeberry juice concentrate was obtained (courtesy of Artemis International (Fort Wayne, IN, USA)) and stored under dark refrigeration (4 °C) conditions. The anthocyanin-rich chokeberry extract was prepared by the methods of Rodriguez-Saona and Wrolstad and He and Giusti [32 ,33 (link)]. Briefly, the juice concentrate was extracted with an equal volume of acetone and filtered through Whatman #1 paper. Hydrophobic compounds were partitioned from the acetone extract by addition of two parts chloroform to one part acetone filtrate (v/v) and storage overnight at 4 °C. The aqueous phase was collected and residual acetone or chloroform was removed by rotary evaporation. The resulting material was then purified by cation exchange (MCX SPE cartridge, Waters Corp., Milford, MA, USA). Neutral and anionic phenolics were removed with 0.1% trifluoroacetic acid in water and then 0.1% trifluoroacetic acid in methanol. Anthocyanins were then eluted into a flask containing 500 µL 88% formic acid with water:methanol 40:60 v/v with 1% NH₄OH followed by methanol with 1% NH₄OH. The anthocyanin content of the extract was determined by High Pressure Liquid Chromatography (HPLC), as described below.

Sample cleanup for LC-MS/MS analysis was performed as follows. The calibration standard, tissue homogenate, cell lysates and cell media samples (100 μL) were transferred into a 1.5-mL eppendorf tube. Then, 10 μL of ISTD working solution and 900 μL of PBS buffer were added. The ADMA was extracted with MCX SPE cartridge (Waters) as described previously^{34 (link)}. After SPE, the only 100ul eluent of final volume of 1 mL was transferred into a new glass tube and evaporated at 40 °C under N2 gas stream. The ADMA derivatization reaction was performed using PITC reagent^{35 (link)}. The residue was reconstituted in 100 μL of coupling solution (ACN:pyridine:triethylamine:water, 10:5:2:3), vortexed for 30 s and added 5 μL of PITC solution. The reaction was allowed to proceed for 5 minutes at room temperature and the samples were dried under N2 at 40 °C. Before analysis the samples were reconstituted in 200 μL of mobile phase. Then, 1 μl sample was injected onto LC-MS/MS system.