Introduction

Preparation of fatty acids from biological materials for GC/MS analysis is typically time consuming, requiring separate extraction, hydrolysis, and derivatization steps. For samples of bacterial biomass, the workup can be combined into a single rapid, quantitative step resulting in FAMEs ready for GC/MS or GC/IRMS analysis. The following procedure is our adaptation of the method published by Rodriguez-Ruiz et al (Biotechnology Techniques, Vol 12, No 9, September 1998, pp. 689–691). Note that the procedure is not recommended for plant materials, where hydrolysis of the more recalcitrant structural carbohydrates is required to get good lipid yields. For those samples, we recommend overnight saponification followed by extraction.

Materials

• anhydrous methanol
• acetyl chloride
• hexane, distilled water
• heating block (100C)
• silica-gel column for cleanup (optional)

Warnings

• Acetyl chloride is highly reactive with substances containing acidic protons, including water, methanol, etc. The reaction is highly exothermic, and can result in rapid boiling and/or explosions of a highly acidic solution. Always wear gloves and safety glasses, and add acetyl chloride to methanol slowly while keeping the mixture cold on ice.
• Capped vials have occasionally burst while being heated, especially in this procedure where high temperatures (100C) are employed. Keep the heating block in the hood with the sash lowered while samples are being heated, and do not let others work in the hood while heating. Wear your safety glasses.

Procedure

1. Samples must be thoroughly dried for this procedure. We typically freeze-dry biomass overnight in the culture tubes used for extraction (note A). Add internal standard as needed.
2. Prepare a mixture of 20:1 v/v anhydrous methanol/acetyl chloride as follows: transfer methanol into a clean 40mL vial, cap, and set to cool on ice for ~5 minutes. Add acetyl chloride dropwise from a pipette, swirling the mixture as you add to prevent boiling. Return to ice frequently to keep cold. Failure to follow this procedure will result in spattering and/or boiling of the mixture, which should be avoided. The mixture needs to remain anhydrous, so limit exposure to air as much as possible.
3. To each sample, add 2 mL of the methylation mixture (above) and 1 mL of hexane. Cap the tube tightly and heat at 100C for 10 minutes (note B). A single phase should form. This volume works well for biomass samples containing <100mg dry weight; scale volume up as needed.
4. Let the sample cool to room temperature. Add 2 mL dionized water and 2 mL hexane. Two phases should form. Remove the hexane (top) phase and collect in a vial. To maximize extraction yield, you can repeat the extraction with additional 2 mL additions of hexane (note C).
5. The hexane extract can now be concentrated into a GC vial and injected directly on the GC/MS. To provide a cleaner FAME sample, we recommend first cleaning up the extract on a short silica-gel column using hexane to elute the FAMEs. This is a useful safeguard because it also prevents inadvertant injection of acidic water into the GC/MS if the extraction was not done carefully.

Notes

1. Biomass can be transferred into culture tubes with distilled water. Depending on how much water is in the culture tubes, they will sometimes break as the water expands when freezing. For small samples (a few mL), laying the tubes at an angle in the freezer is usually sufficient. For larger volumes of water, we recommend freezing by swirling the culture tube in a dewar of liquid N2, slowly lowering the tube so that the water freezes from the bottom up. Once fully frozen, you can transfer to the freezer for storage.
2. We are heating the reaction mixture to well above its boiling point. If the cap is not perfectly sealed, the solvent will all evaporate during the 10 minute heating time. We typically use Teflon capliners, but still occasionally have tubes leak. Check the tubes after several minutes, and recap any that appear to be leaking.
3. If you shake the mixture vigorously after adding hexane, many samples will produce a dense emulsion that is hard to separate. The easiest approach is to avoid vigorous shaking in the first place. If you do get an emulsion, spinning down the sample in a centrifuge, or else just being really patient, are your two best options.