TITLE:  Extraction of Oxycodone from Rat Brain for Mass Spec Analysis
AUTHORS: Sierra Simpson, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Extraction of Oxycodone from Rat Brain for Mass Spec Analysis</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:center">Adapted from Momper Lab protocol</div></div></div>

[STEPS]
?. [Brain Homogenization]
Brains are snap-frozen upon harvest and are stored in -80C until ready for processing.Thaw out the tissue slowly on ice for 5-10 minutes, once thawed place the brain on a sterile dish and mince the entire brain using a fresh razor blade.
?. [Brain Homogenization]
Add ~20- 40mg minced tissue to each homogenizing tube.
?. [Brain Homogenization]
Add ~375ul 4C acetonitrile (ACN) to each homogenizing tube containing the tissue and beads.  0.053mg tissue/μl ACN (example: 20mg tissue / 375 μl ACN)
?. [Brain Homogenization]
Add internal standard d6-Oxycodone to a concentration of 0.4ng/ul ( Cayman chemicals # 19249)
?. [Brain Homogenization]
Store these tubes on ice until ready to homogenize on a bead beater machine.
?. [Brain Homogenization]
Put tubes in a bead beater machine to homogenize and break up the tissue(s).
?. [Bead Beating ]
The bead beater machine is located in the SKAGGS building in the Palmer Taylor lab / Dorrestein Labs. Make sure to contact them before use to reserve time on the machine. Male sure the sample tubes are secured – this machine shakes the samples quite quickly and if not properly secured can cause severe damage to the machine and the lab.
?. [Bead Beating ]
Set the machine at 25,000 cycles per min.
?. [Bead Beating ]
Set the vibration time for 30 seconds
?. [Bead Beating ]
After 30 seconds of vibration wait for 30 seconds with no vibration (prevents heat buildup of the tissue from the vibrating beads)
?. [Bead Beating ]
Repeat the vibration step two more times (total of 3 cycles)
?. [Bead Beating ]
Transfer all the samples back on ice and allow to cool for 5 – 10 mins.
?. [Bead Beating ]
After 5 – 10 mins centrifuge the homogenizing tubes at 5000 rpm for 5 mins.
?. [Bead Beating ]
Transfer the clear supernatant to the pre-labeled 1.5ml epitube and place on ice (~500μl):
?. [Bead Beating ]
Place the homogenizing tube back on ice until ready to add H2O to wash along the wall of the homogenizing tube to collect any remaining sample stuck along the wall of the tube.
?. [Bead Beating ]
Add ~125μl H2O (=0.160mg/μl H2O, example 20mg tissue/125μl H2O) to the homogenizing tube.
?. [Bead Beating ]
Vortex vigorously for 3-5 seconds.
?. [Bead Beating ]
Transfer the resulting H2O mix (~125μl) to the pre-labeled 1.5ml polypropylene bullet tube containing ~500μl of homogenized supernatant – at this point the clear solution will become cloudy and place on ice (~625μl)
?. [Bead Beating ]
Centrifuge samples at 5000 rpm for 5 mins.
?. [Filtration and Prep for Mass Spec ]
Transfer the clear supernatant (~625μl) to a 1cc syringe with an attached filter unit (Millex – GV, catalog # SLGVR04NL) - this step further cleans the solution to filter out any additional contaminants that can clog the mass spec.
?. [Filtration and Prep for Mass Spec ]
Discard the tube containing the pellet
?. [Filtration and Prep for Mass Spec ]
After transferring the clear supernatant to the 1cc syringe, insert the plunger of the syringe to the 1cc syringe to elute the solution through the filter unit into a pre-labeled mass spec vial.
?. [Filtration and Prep for Mass Spec ]
Place a cap on the mass spec vial containing the filtered extracted tissue. The sample is ready for mass spec analysis (store the samples in -20C until ready for mass spec analysis, but we prefer to extract and run the sample in the same day if possible)