||Homogenized tissue is hydrolized with HCl in tubes incubated on a heating block at 110 to 120 degrees celcius for 2 hours before cooling the mixture to ambient temperature. The pH of the hydrolysate is adjusted to 9.5 to 9.70 using NaOH. Sodium chloride
is added to the tubes followed by ethyl acetate prior to extraction by repeated centrifugation. The ethyl acetate extract is evaporated to dryness, reconstituted in hexane and mixed by vortex prior to cleane-up using a silica cartridge. The centrifuge tube
is rinsed with 30 percent ethyl acetate,hexane and the contents loaded onto the cleanup column as well. Ethyl acetate is further used to wash the column contents into waste. The tubes are further rinsed twice with sodium phosphate and both eluates combined.
After refrigerating the contents overnight at 2-8 degrees celcius, the aqueous sodium phosphate solution is further cleaned-up using C18 cartridge material. After loading the extract, this cartridge is washed with water and 5 percent MeOH,H2O (v,v) into waste
before eluting the contents from the cartridge with 40 percent MeOH,H2O (v,v). This eluate is loaded onto SCX cartridges for final cleanup. After washing the column with methanol, the analyte is eluted using 10 percent NH4OH,MeOH. This solution is evaporated
to dryness and the residue reconstituted in methanol. An aliquot for LCMSMS (the rest may be used for HPLC analysis) is evaporated to dryness and after storage (capped) in a freezer at minus ten degree celcius, the aliquot is later reconstituted in the mobile
phase of 0.005 M Tetrabutyl Ammonium Phosphate,Methanol (46.5,53.3 v,v) and pressed through a 0.2 micron filter ready for analysis by LC,ESI-MS,MS. A Zorbax Rx C8 analytical column is used for separation.|