Bligh Dyer Extraction - tromsdahl/personal-site GitHub Wiki

Background

The Bligh-Dyer extraction is a biphasic liquid-liquid extraction using methanol, water, and chloroform. It has been regarded as the gold standard for lipid extractions. The method was first published by E. G. Bligh and W. J. Dyer in 1959. Below are the step-by-step instructions for performing a Bligh-Dyer extraction for serum/plasma/cells or tissue.

Serum/Plasma/Cell Extraction

Sample Processing at Client’s Lab

Plasma and sera samples should be snap frozen and stored at -80°C until ready to submit. Cells should be pelleted by centrifugation, supernatant removed, and then washed once with PBS. Pellet cells again by centrifugation and remove the PBS wash. Snap freeze cell pellets and store at -80°C until ready to submit.

A) Solution and Internal Standards Preparation

  1. CHCl3/MeOH (1:1, v/v): Combine 50 mL of chloroform (CHCl3) with 50 mL of methanol (MeOH).
  2. Polar metabolite IS mix: Combine 300 μL of each of the IS mixes below into a single 12 x 75 mm glass tube. Vortex to mix.
    • Carnitine/Acylcarnitine Standard Mix Set B (Cambridge Isotope Laboratories, ca. no. NSK-B-1)
    • Carnitine/Acylcarnitine Standard Mix Supplement to NSK-B (Cambridge Isotope Laboratories, ca. no. NSK-B-G1)
    • Metabolomics QC Kit (Cambridge Isotope Laboratories, ca. no. MSK-QC-KIT)
    • Organic Acid Mix (Cambridge Isotope Laboratories, ca. no. MSK-OA-1)
  3. 1:10 diluted lipid IS mix: Combine 100 μL of each of the IS mixes below with 800 μL of CHCl$_3$/MeOH (1:1, v/v) into an amber autosampler vial. Vortex to mix.
    • UltimateSPLASH ONE IS (Avanti Polar Lipids, ca. no. 330820)
    • SphingoSPLASH I (Avanti Polar Lipids, ca. no. 330734)
  4. 5% SDS, 50 mM TEAB, pH 7.1: Combine 2.5 mL of 1 M TEAB with 22.5 mL of LCMS grade water, then pH to 7.1 using 1 N HCl. Finally, add 25 mL of 10% SDS.

B) Sample Extraction

  1. Aliquot or transfer samples into 1.5 mL microcentrifuge tubes.
    • Plasma and serum: aliquot 25 μL
    • Cells: add 50 μL of LC-MS grade water to resuspend cell pellet
  2. Prepare an “Extracted IS” control by aliquoting 50 μL of LC-MS grade water into an empty 1.5 mL microcentrifuge tube.
  3. Prepare an “Extraction Blank” control by aliquoting 50 μL of LC-MS grade water into an empty 1.5 mL microcentrifuge tube.
  4. Add 10 μL of Polar metabolite IS mix to samples and “Extracted IS” control.
    • Note: Do NOT add IS to the "Extraction Blank" control.
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