Laboratory protocol for hydrolysis of lipids [6]:

A solution of 0.1M potassium hydroxide in 90% aqueous ethanol is prepared immediately before use by adding 1 volume of standard 1M aqueous potassium hydroxide to 9 volumes of ethanol. To the lipid sample (up to 6 mg) in a stoppered centrifuge tube is added 0.1M potassium hydroxide in 90% aqueous ethanol (0.25 mL per mg sample). Leave at 50°C in a water bath or heating block for 3 hours. Remove from water bath and add 0.2M hydrochloric acid (0.05 mL per mg sample) using a Pasteur pipette. Add isohexane (3 mL), diethyl ether (3 mL) and distilled water (2 mL). Shake thoroughly and, if necessary (if there are not two distinct layers and/or an emulsion is formed), centrifuge for 5 min (about 400 rpm). Put upper organic layer through a short (3 cm) column of anhydrous sodium sulfate (prepared in a Pasteur pipette, plugged with a small piece of cotton wool, and pre-washed with 3 mL isohexane-diethyl ether (1:1, by vol.) prior to use) to dry. Collect in a test tube. Repeat extraction of lower aqueous layer with isohexane-diethyl ether (3 mL, 1:1, by vol.) and put the organic layer through the sodium sulfate column. Wash sample through the column into the tube with more isohexane-diethyl ether (3 mL). Take sample to dryness in a gentle stream of nitrogen on a heating block at 30°C.

Laboratory protocol for preparation of picolinyl esters via an imidazole intermediate [1]:

The reaction, as detailed below, is moisture sensitive and all reagents should be protected from moisture (i.e. by storing in a the presence of a desiccant). The carbonyldiimidazole solution, in particular, should be freshly made up each time a set of samples are derivatized. The picolinyl derivatizing reagent is prepared by dissolving 3-(hydroxymethyl)pyridine (900 µL) in dichloromethane (9 mL) and triethylamine (9 mL), and adding a little anhydrous sodium sulfate (2-3 mm in tube). It can be used for up to one month if kept in a sealed tube (with PTFE-lined screw top) in the refrigerator. The free fatty acid sample (up to 1 mg), in a centrifuge tube, is dissolved in dichloromethane (100 µL) and a solution of 1,1'-carbonyldiimidazole (100 µL, 10 mg/mL in dichloromethane, freshly prepared) is added. Leave 1 min (not longer) at room temperature before proceeding to the next step. Add picolinyl reagent (200 µL). Agitate and leave for 10 min. at 37°C. At the end of this time add acetic acid (25 µL). Take to dryness on a heating block at 30°C with a gentle stream of nitrogen. Add isohexane (5 mL) and distilled water (2 mL). Shake thoroughly and vortex. Put upper isohexane layer through a short (4 cm) column of anhydrous sodium sulfate in a Pasteur pipette (with a small cotton wool plug and pre-washed with isohexane) to dry. Re-extract the aqueous layer with fresh isohexane (2 mL), and pass through the column also. If necessary, a little crystalline potassium chloride can be added to break emulsions. Finally, wash through column with isohexane (1 mL) and evaporate the combined isohexane eluents. Dissolve sample in an appropriate amount of isohexane for GC and GC-MS analyses.

Laboratory protocol for preparation of picolinyl esters via transesterification [4]:

A solution of potassium tert-butoxide in tetrahydrofuran (0.1 mL, 1.0 M) is added to 3-pyridylcarbinol (0.2 mL). After mixing, the lipid sample (up to 10 mg) in dry dichloromethane (1 mL) is added, and the mixture is held at 40°C for 30 min in a closed vial. After cooling to room temperature, water (2 mL) and hexane (4 mL) are added, and the organic phase is collected, dried over anhydrous sodium sulfate, and evaporated. The sample is dissolved in isohexane for GC-MS analysis.

Laboratory protocol for preparation of picolinyl esters via acid chlorides [2]:

All reactions are carried out in 0.3 ml screw cap vials. The acid (20 μg) is heated with thionyl chloride (20 μL) for 10 min. at 100ºC. The thionyl chloride is evaporated in a stream of nitrogen, and a solution of 20% 3-(hydroxymethyl)pyridine in acetonitrile (10 μL) is added, and the mixture heated for 1 min. at 100ºC. An aliquot is injected directly onto the GC column.

Laboratory protocol for preparation of DMOX derivatives by the one-step reaction [5]:

To the lipid sample (up to 2 mg) in a test tube is added 2-amino-2-methyl-1-propanol (0.25 g). The vessel is flushed with nitrogen, stoppered, and placed in a heating block, at 190ºC overnight. Flush the tube with nitrogen when the tube is up to temperature to help to eliminate moisture and minimize autoxidation. A little glycerol added to the well of the heating block ensures good contact and that the correct temperature is maintained. Next day, remove the test tube from the heating block, allow to cool to room temperature and wash off any glycerol on the external surface with water. Add diethyl ether-isohexane (1:1, v/v; 5 mL) to the tube, washing down the internal surface, followed by water (5 mL). Shake thoroughly and allow layers to settle. If necessary, addition of a little sodium or potassium chloride will usually break up any interfacial layers. Transfer the organic layer with a pasteur pipette to a fresh test-tube, and re-extract the aqueous layer with fresh solvent (2 mL). Add distilled water (3 mL) to the combined solvent layers, shake and allow to settle. Transfer the solvent layer to a fresh tube with a pasteur pipette and add anhydrous sodium sulfate (about 1 cm). Leave for one hour with occasional vigorous shaking, then pass solvent layer through a short (3 cm) column of anhydrous sodium sulfate (prepared in a Pasteur pipette, plugged with a small piece of cotton wool, and pre-washed with ca. 3 mL isohexane prior to use) to dry, into a fresh test tube. Wash sample from the sodium sulfate tube through column with isohexane (2 mL). Take sample to dryness in a gentle stream of nitrogen on a heating block at 30ºC. Dissolve the sample in an appropriate amount of isohexane for GC and GC-MS analyses. Adding a few crystals of anhydrous sodium sulfate stabilizes the derivative on storage.

Note that thorough drying in this way gives a more stable product. All steps should be carried out as expeditiously as possible. (Note that DMOX derivatives are not easily purified by adsorption chromatography).

Laboratory protocol for preparation of DMOX derivatives by the two step reaction [8]:

The fatty acid must first be converted to the acid chloride as in the method for preparation of picolinyl esters via the acid chloride. This is used immediately for the preparation of the required derivatives.

A solution of 2-amino-2-methylpropanol in dichloromethane (10 mg/mL) is prepared and stored over anhydrous sodium sulfate. It is stable in the refrigerator for about 1 month. This solution (0.5 mL) is added to the freshly prepared acid chloride, cooled in an ice bath, and the mixture is then left to warm up to room temperature for an hour. The solvent is removed in a stream of nitrogen, and trifluoroacetic anhydride (0.5 mL) is added. After 1 hour on a heating block at 50ºC, or 3 hours at room temperature, the reagent is removed in a stream of nitrogen. Isohexane (5 mL) is added followed by water (2 mL), and the product is obtained as in the previous reaction, taking care to dry the product thoroughly.

Note that compounds that appear to be closely related to the DMOX derivatives but eluting a little later on GC can be 1-2% of the total, and at the moment we have no method of eliminating these.

Laboratory protocol for preparation of pyrrolidide derivatives from fatty acid methyl esters [12]:

The fatty acid methyl ester (up to 10 mg) is dissolved in freshly distilled pyrrolidine (1 mL), acetic acid (0.1 mL) is added, and the mixture is heated at 100ºC for 1 hour. Excess pyrrolidine is blown off in a stream of nitrogen at 50ºC, and then the residue is taken up in hexane-diethyl ether (1:1, v/v; 8 mL) and is washed three times with water (4 mL portions). After drying over anhydrous sodium sulfate, the required product is obtained on evaporation of the solvent.