TITLE: BAF_Protocol_009 Metabolomics: Lipid Extraction 
AUTHORS: Nicholas Sherman

[DESCRIPTION]
This protocol uses a similar method to Protocol_008 but retains the chloroform phase containing lipids and other very hydrophobic molecules. Extractions are done in a way to make sure the final sample is mostly lipids. This type of sample will need to be run on a different column using different buffers from those used for the aqueous/methanol metabolites.

[GUIDELINES]
The dry down of this phase is best done using a stream of inert nitrogen to minimize oxidation of lipids and directly before analysis.

[STEPS]
SECTION: Liquid Samples: Urine, Plasma, Culture Media
1. To each sample containing 100 uL add 750 µL of -20°C cold Chloroform:methanol (2:1) mixture and vortex
SECTION: Liquid Samples: Urine, Plasma, Culture Media
2. Shake tubes vigorously for 30 min at 4°C in temperature temperature-controlled thermal shaker
SECTION: Liquid Samples: Urine, Plasma, Culture Media
3. Add 400 µL of water, shake vigorously, and centrifuge for 10 min at 10000 rpm for phase separation
SECTION: Liquid Samples: Urine, Plasma, Culture Media
4. Discard the top aq. methanolic phase (if interested in soluble metabolite, recover this phase. See BAF_Protocol_008)
SECTION: Liquid Samples: Urine, Plasma, Culture Media
5. Recover the Lower phase to new clean Eppendorf
SECTION: Liquid Samples: Urine, Plasma, Culture Media
6. To each tube with 500 µL of chloroform phase, add 500 µL of cold Chloroform:methanol (2:1) mixture
SECTION: Liquid Samples: Urine, Plasma, Culture Media
7. Vortex and shaken vigorously for 30 min at 4°C in temperature controlled thermal shaker
SECTION: Liquid Samples: Urine, Plasma, Culture Media
8. Add 200 µL of water, shake vigorously, centrifuge for 10 min at 10000 rpm and recover the lower organic phase as a lipid mixture in glass tubes and store at -80°C
SECTION: Liquid Samples: Urine, Plasma, Culture Media
9. Before running, add 10 µL of Avanti Splash Lipidomix to each sample as internal standard and dry samples under gentle stream of N2 using a Recti-VapTM Evaporator (Thermo Fisher Scientific) at 40°C
SECTION: Liquid Samples: Urine, Plasma, Culture Media
10. Reconstitue in 110 µL of methanol:isoproponal (1:1)
SECTION: Liquid Samples: Urine, Plasma, Culture Media
11. Transfer 100 uL to borosilicate glass inserts kept inside a screw-capped glass autosampler vials (Agilent)
SECTION: Solid samples: Cell Media, Stool, Tissue
12. Place the sample in reinforced tubes: Frozen tissue slice or lyophilized stools. For cell pellets – mix well with 50-100 uL of water transfer solution and suspended cells to reinforced tubes
SECTION: Solid samples: Cell Media, Stool, Tissue
13. To each sample add 5 stainless steel balls, 750 µL of -20°C cold Chloroform:methanol (2:1) mixture.
Disrupt cells/tissues with Fisher Bead Mill 24 (speed: 5m/s, time: 20 sec, number of cycle: 3, dwell/pause between runs: 10 sec).
SECTION: Solid samples: Cell Media, Stool, Tissue
14. Shake tubes vigorously for 30 min at 4°C in temperature temperature-controlled thermal shaker
SECTION: Solid samples: Cell Media, Stool, Tissue
15. Add 400 µL of water, shake vigorously, and centrifuge for 10 min at 10000 rpm for phase separation. Discard the top aq. methanolic phase (if interested in soluble metabolite, recover this phase. See BAF_Protocol_08)
SECTION: Solid samples: Cell Media, Stool, Tissue
16. Recover the Lower phase to new clean Eppendorf - extract lipids. Perform steps 06-11 from the above protocol