Animal and Human Metabolite Preparation Protocol

Please read Metabolite Processing Precautions before proceeding.

⦁ Prepare and separate all material and solutions before you start. Check the Recommended Resources List.

⦁ Allow the samples thaw in ice.

⦁ While samples are thawing, label two tubes, one as QCp and the other as QCnp (pooled quality control). NOTE: One tube will be for the polar (QCp) fraction and the other for the non-polar fraction (QCnp).

⦁ Pulverize the frozen tissue using a cryomill (recommended) or a LN₂ precooled mortar and pestle.

⦁ Lyophilize pulverized tissue sample overnight.

⦁ Weigh 20 mg from the homogenized dried tissue powder into a precooled 2mL cryotube.

⦁ Prepare two tubes labeled as QC (pooled quality control). NOTE: One tube will be for the polar (QCp) fraction nd the other for the non-polar fraction (QCnp). If only one LC method is used, you may combine the fractions thus using only one QC tube.

⦁ Add 700 μL of ice-cold extraction solution C (Polar extraction).

⦁ Add 80 mg of zirconium beads, beat for 10 s and cool down on ice for 30 s. Repeat the cycle 3 more times.

⦁ Centrifuge the samples at 10,000 g for 10 min at 4°C.

⦁ Collect 80 μL of the supernatant and transfer to the QCp tube.

⦁ Collect the remaining supernatant in a new labeled tube (polar fraction). NOTE: make aliquots of 200 µL.

⦁ Place the tubes containing the polar fraction in dry ice.

⦁ IMMEDIATELY after collection of the polar fraction, add 700 μL of ice-cold extraction solution D (Non-polar extraction).

⦁ Repeat the homogenization cycle and centrifuge as previously described.

⦁ Collect 80 μL of the supernatant and transfer to the QCnp tube.

⦁ Collect the supernatant and transfer it to a new labeled glass tube (non-polar fraction). NOTE: make aliquots of 200 µL.

⦁ Freeze-dry the polar fraction in a lypholyzer.

⦁ Freeze-dry the non-polar extract in the glass cupboard (lypholyzers are NOT compatible with chloroform).

PAUSE POINT Store the samples at -80°C for up to 12 weeks.